Benzoimidazoles as prolyl hydroxylase inhibitors

ABSTRACT

Certain Benzoimidazole compounds are described, which are useful as prolyl hydroxylase inhibitors. Such compounds may be used in pharmaceutical compositions and methods for the treatment of disease states, disorders, and conditions mediated by prolyl hydroxylase activity. Thus, the compounds may be administered to treat, e.g., Anemia, vascular disorders, metabolic disorders, and wound healing.

FIELD OF THE INVENTION

The present invention relates to certain benzoimidazole compounds,pharmaceutical compositions containing them, and methods of using themfor the treatment of disease states, disorders, and conditions mediatedby prolyl hydroxylase activity.

BACKGROUND OF THE INVENTION

Cells respond to hypoxia by activating the transcription of genesinvolved in cell survival, oxygen delivery and utilization,angiogenesis, cellular metabolism, regulation of blood pressure,hematopoiesis, and tissue preservation. Hypoxia-inducible factors (HIFs)are key transcriptional regulators of these genes (Semenza et al., 1992,Mol Cell Biol., 12(12):5447-54; Wang et al., 1993, J Biol Chem.,268(29):21513-18; Wang et al., 1993, Proc Natl Acad Sci., 90:4304-08;Wang et al., 1995, J Biol Chem., 270(3):1230-37). Three forms of HIF-αhave been described: HIF-1α, HIF-2α and HIF-3α (Scheuermann et al.,2007, Methods Enzymol., 435:3-24). Pairing of a HIFα sub-unit withHIF-1β forms a functional heterodimeric protein that subsequentlyrecruits other transcriptional factors such as p300 and CBP (Semenza,2001, Trends Mol Med., 7(8):345-50).

A family of highly conserved oxygen, iron, and 2-oxoglutarate-dependentprolyl hydroxylase (PHD) enzymes mediate the cells response to hypoxiavia post-translational modification of HIF (Ivan et al., 2001, Science,292:464-68; Jaakkola et al., 2001, Science, 292:468-72). Under normoxicconditions, PHD catalyzes the hydroxylation of two conserved prolineresidues within HIF. Von Hippel Lindau (VHL) protein binds selectivelyto hydroxylated HIF. The binding of VHL renders HIF a target forpolyubiquitination by the E3 ubiquitin ligase complex and its subsequentdegradation by the 26S proteasome (Ke et al., 2006, Mol Pharmacol.70(5):1469-80; Semenza, Sci STKE., 2007, 407(cm8):1-3). As the affinityof PHD for oxygen is within the physiological range of oxygen and oxygenis a necessary co-factor for the reaction, PHD is inactivated whenoxygen tension is reduced. In this way, HIF is rapidly degraded undernormoxic conditions but accumulates in cells under hypoxic conditions orwhen PHD is inhibited.

Four isotypes of PHD have been described: PHD1, PHD2, PHD3, and PHD4(Epstein et al., 2001, Cell, 107:43-54; Kaelin, 2005, Annu Rev Biochem.,74:115-28; Schmid et al., 2004, J Cell Mol Med., 8:423-31). Thedifferent isotypes are ubiquitously expressed but are differentiallyregulated and have distinct physiological roles in the cellular responseto hypoxia. There is evidence that the various isotypes have differentselectivity for the three different HIFα sub-types (Epstein et al.,supra). In terms of cellular localization, PHD1 is primarily nuclear,PHD2 is primarily cytoplasmic, and PHD3 appears to be both cytoplasmicand nuclear (Metzen E, et al. 2003, J Cell Sci., 116(7):1319-26). PHD2appears to be the predominant HIFα prolyl hydroxylase under normoxicconditions (Ivan et al., 2002. Proc Natl Acad Sci. USA, 99(21):13459-64;Berra et al., 2003, EMBO J., 22:4082-90). The three isotypes have a highdegree of amino-acid homology and the active site of the enzyme ishighly conserved.

The HIF target gene products are involved in a number of physiologicaland pathophysiological processes including but not limited to:erythropoiesis, angiogenesis, regulation of energy metabolism, vasomotorfunction, and cell apoptosis/proliferation. The first gene described asa HIF target was that encoding erythropoietin (EPO) (Wang et al., 1993,supra). It was recognized that a reduction in the oxygen carryingcapacity of the blood is sensed in the kidney and that the kidney andliver respond by releasing more EPO, the hormone that stimulates redblood cell proliferation and maturation. EPO has a number of otherimportant effects on non-hematopoietic cell types and has emerged as akey tissue-protective cytokine (Arcasoy, 2008, Br J Haematol.,141:14-31). Thus EPO is now implicated in wound healing and angiogenesisas well as the response of tissues to ischemic insult. Most of theenzymes involved in anaerobic glycolysis are encoded by HIF target genesand as a result glycolysis is increased in hypoxic tissues (Shaw, 2006,Curr Opin Cell Biol., 18(6):598-608). The known HIF target gene productsin this pathway include but are not limited to: glucose transporterssuch as GLUT-1 (Ebert et al., 1995, J Biol Chem., 270(49):29083-89),enzymes involved in the break down of glucose to pyruvate such ashexokinase and phosphoglycerate kinase 1 (Firth et al., 1994, Proc NatlAcad Sci. USA, 91:6496-6500) as well as lactate dehydrogenase (Firth etal., supra). HIF target gene products are also involved in theregulation of cellular metabolism. For example, pyruvate dehydrogenasekinase-1 is a target HIF gene product and regulates the entry ofpyruvate into the Kreb's cycle by reducing the activity of pyruvatedehydrogenase by phosphorylation (Kim et al., 2006, Cell Metab.,3:177-85; Papandreou et al., 2006, Cell Metab., 3:187-197). HIF targetgene products are also involved in angiogenesis. For example, vascularendothelial growth factor (VEGF) (Liu et al., 1995, Circ Res.,77(3):638-43) is a known regulator of angiogenesis and vasculogenesis.HIF target gene products also function in the regulation of vasculartone and include heme oxygenase-1 (Lee et al., 1997, J Biol Chem.,272(9):5375-81). A number of HIF regulated gene products such asplatelet-derived growth factor (PDGF) (Yoshida et al., 2006, JNeurooncol., 76(1):13-21), vascular endothelial growth factor (Breen,2007, J Cell Biochem., 102(6):1358-67) and EPO (Arcasoy, supra) alsofunction in the coordinated response to wound healing.

Targeted disruption of the prolyl hydroxylase (PHD) enzyme activity bysmall molecules has potential utility in the treatment of disorders ofoxygen sensing and distribution. Examples include but are not limitedto: anemia; sickle cell anemia; peripheral vascular disease; coronaryartery disease; heart failure; protection of tissue from ischemia inconditions such as myocardial ischemia, myocardial infarction andstroke; preservation of organs for transplant; treatment of tissueischemia by regulating and/or restoring blood flow, oxygen deliveryand/or energy utilization; acceleration of wound healing particularly indiabetic and aged patients. In addition, targeted disruption of PHD isexpected to have utility in treating metabolic disorders such asdiabetes and obesity.

HIF has been shown to be the primary transcriptional factor that leadsto increased erythropoietin production under conditions of hypoxia (Wanget al., 1993, supra). While treatment with recombinant humanerythropoietin has been demonstrated to be an effective method oftreating anemia, small molecule mediated PHD inhibition can be expectedto offer advantages over treatment with erythropoietin. Specifically,the function of other HIF gene products are necessary for hematopoesisand regulation of these factors increases the efficiency ofhematopoesis. Examples of HIF target gene products that are critical forhematopoesis include: transferrin (Rolfs et al., 1997, J Biol Chem.,272(32):20055-62), transferrin receptor (Lok et al., 1999, J Biol Chem.,274(34):24147-52; Tacchini et al., 1999, J Biol Chem., 274(34):24142-46)and ceruloplasmin (Mukhopadhyay et al., 2000, J Biol Chem.,275(28):21048-54). Hepcidin expression is also suppressed by HIF(Peyssonnaux et al., 2007, J Clin Invest., 117(7):1926-32) and smallmolecule inhibitors of PHD have been shown to reduce hepcidin production(Braliou et al., 2008, J Hepatol., 48:801-10). Hepcidin is a negativeregulator of the availability of the iron that is necessary forhematopoesis, so a reduction in hepcidin production is expected to bebeneficial to the treatment of anemia. PHD inhibition may also be usefulwhen used in conjunction with other treatments for anemia including ironsupplementation and/or exogenous erythropoietin. Studies of mutations inthe PHD2 gene occurring naturally in the human population providefurther evidence for the use of PHD inhibitors to treat anemia. Tworecent reports have shown that patients with dysfunctional mutations inthe PHD2 gene display increased erythrocytosis and elevated bloodhemoglobin (Percy et al., 2007, PNAS, 103(3):654-59; A1-Sheikh et al.,2008, Blood Cells Mol Dis., 40:160-65). In addition, a small moleculePHD inhibitor has been evaluated in healthy volunteers and patients withchronic kidney disease (U.S. pat. appl. US2006/0276477, Dec. 7, 2006).Plasma erythropoietin was increased in a dose-dependent fashion andblood hemoglobin concentrations were increased in the chronic kidneydisease patients.

Metabolic adaptation and preservation of tissues are jeopardized byischemia. PHD inhibitors increase the expression of genes that lead tochanges in metabolism that are beneficial under ischemic conditions(Semenza, 2007, Biochem J., 405:1-9). Many of the genes encoding enzymesinvolved in anaerobic glycolysis are regulated by HIF and glycolysis isincreased by inhibiting PHD (Shaw, supra). Known HIF target genes inthis pathway include but are not limited to: GLUT-1 (Ebert et al.,supra), hexokinase, phosphoglycerate kinase 1, lactate dehydrogenase(Firth et al., supra), pyruvate dehydrogenase kinase-1 (Kim et al.,supra; Papandreou et al., supra). Pyruvate dehydrogenase kinase-1suppresses the entry of pyruvate into the Kreb's cycle. HIF mediates aswitch in the expression of the cytochromes involved in electrontransport in the mitochondria (Fukuda et al., 2007, Cell,129(1):111-22). This change in the cytochrome composition optimizes theefficiency in ATP production under hypoxic conditions and reduces theproduction of injurious oxidative phosphorylation by-products such ashydrogen peroxide and superoxide. With prolonged exposure to hypoxia,HIF drives autophagy of the mitochondria resulting a reduction in theirnumber (Zhang H et al., 2008, J Biol Chem., February 15 Epub ahead ofprint). This adaptation to chronic hypoxia reduces the production ofhydrogen peroxide and superoxide while the cell relies on glycolysis toproduce energy. A further adaptive response produced by HIF elevation isup-regulation of cell survival factors. These factors include:Insulin-like growth factor (IGF) 2, IGF-binding protein 2 and 3 (Feldseret al., 1999, Cancer Res. 59:3915-18). Overall accumulation of HIF underhypoxic conditions governs an adaptive up-regulation of glycolysis, areduction in oxidative phosphorylation resulting in a reduction in theproduction of hydrogen peroxide and superoxide, optimization ofoxidative phosphorylation protecting cells against ischemic damage.Thus, PHD inhibitors are expected to be useful in organ and tissuetransplant preservation (Bernhardt et al., 2007, Methods Enzymol.,435:221-45). While benefit may be achieved by administering PHDinhibitors before harvesting organs for transplant, administration of aninhibitor to the organ/tissue after harvest, either in storage (e.g.,cardioplegia solution) or post-transplant, may also be of therapeuticbenefit.

PHD inhibitors are expected to be effective in preserving tissue fromregional ischemia and/or hypoxia. This includes ischemia/hypoxiaassociated with inter alia: angina, myocardial ischemia, stroke,ischemia of skeletal muscle. There are a number of lines of experimentalevidence that support the concept that PHD inhibition and subsequentelevation of HIF as a useful method for preserving ischemic tissue.Recently, ischemic pre-conditioning has been demonstrated to be aHIF-dependent phenomenon (Cai et al., 2008, Cardiovasc Res.,77(3):463-70). Ischemic pre-conditioning is a well known phenomenonwhereby short periods of hypoxia and/or ischemia protect tissue fromsubsequent longer periods of ischemia (Murry et al., 1986, Circulation,1986 74(5):1124-36; Das et al., 2008, IUBMB Life, 60(4):199-203).Ischemic pre-conditioning is known to occur in humans as well asexperimental animals (Darling et al., 2007, Basic Res Cardiol.,102(3):274-8; Kojima I et al., 2007, J Am Soc Nephrol., 18:1218-26).While the concept of pre-conditioning is best known for its protectiveeffects in the heart, it also applies to other tissues including but notlimited to: liver, skeletal muscle, liver, lung, kidney, intestine andbrain (Pasupathy et al., 2005, Eur J Vasc Endovasc Surg., 29:106-15;Mallick et al., 2004, Dig Dis Sci., 49(9):1359-77). Experimentalevidence for the tissue protective effects of PHD inhibition andelevation of HIF have been obtained in a number of animal modelsincluding: germ-line knock out of PHD1 which conferred protection of theskeletal muscle from ischemic insult (Aragonés et al., 2008, Nat Genet.,40(2):170-80), silencing of PHD2 through the use of siRNA whichprotected the heart from ischemic insult (Natarajan et al., 2006, CircRes., 98(1):133-40), inhibition of PHD by administering carbon monoxidewhich protected the myocardium from ischemic injury (Chin et al., 2007,Proc Natl Acad Sci. U.S.A., 104(12):5109-14), hypoxia in the brain whichincreased the tolerance to ischemia (Bernaudin et al., 2002, J CerebBlood Flow Metab., 22(4):393-403). In addition, small moleculeinhibitors of PHD protect the brain in experimental stroke models(Siddiq et al., 2005, J Biol Chem., 280(50):41732-43). Moreover, HIFup-regulation has also been shown to protect the heart of diabetic mice,where outcomes are generally worse (Natarajan et a., 2008, J CardiovascPharmacol., 51(2):178-187). The tissue protective effects may also beobserved in Buerger's disease, Raynaud's disease, and acrocyanosis.

The reduced reliance on aerobic metabolism via the Kreb's cycle in themitochondria and an increased reliance on anaerobic glycolysis producedby PHD inhibition may have beneficial effects in normoxic tissues. It isimportant to note that PHD inhibition has also been shown to elevate HIFunder normoxic conditions. Thus, PHD inhibition produces a pseudohypoxiaassociated with the hypoxic response being initiated through HIF butwith tissue oxygenation remaining normal. The alteration of metabolismproduced by PHD inhibition can also be expected to provide a treatmentparadigm for diabetes, obesity and related disorders, includingco-morbidities.

Globally, the collection of gene expression changes produced by PHDinhibition reduce the amount of energy generated per unit of glucose andwill stimulate the body to burn more fat to maintain energy balance. Themechanisms for the increase in glycolysis are discussed above. Otherobservations link the hypoxic response to effects that are expected tobe beneficial for the treatment of diabetes and obesity. Thus, highaltitude training is well known to reduce body fat (Armellini et al.,1997, Horm Metab Res., 29(9):458-61). Hypoxia and hypoxia mimetics suchas desferrioxamine have been shown to prevent adipocyte differentiation(Lin et al., 2006, J Biol Chem., 281(41):30678-83; Carrière et al.,2004, J Biol Chem., 279(39):40462-69). The effect is reversible uponreturning to normoxic conditions. Inhibition of PHD activity during theinitial stages of adipogenesis inhibits the formation of new adipocytes(Floyd et al., 2007, J Cell Biochem., 101:1545-57). Hypoxia, cobaltchloride and desferrioxamine elevated HIF and inhibited PPAR gamma 2nuclear hormone receptor transcription (Yun et al., 2002, Dev Cell.,2:331-41). As PPAR gamma 2 is an important signal for adipocytedifferentiation, PHD inhibition can be expected to inhibit adipocytedifferentiation. These effects were shown to be mediated by theHIF-regulated gene DEC1/Stra13 (Yun et al., supra).

Small molecular inhibitors of PHD have been demonstrated to havebeneficial effects in animal models of diabetes and obesity (Intl. Pat.Appl. Publ. WO2004/052284, Jun. 24, 2004; WO2004/052285, Jun. 24, 2004).Among the effects demonstrated for PHD inhibitors in mouse diet-inducedobesity, db/db mouse and Zucker fa/fa rat models were lowering of: bloodglucose concentration, fat mass in both abdominal and visceral fat pads,hemoglobin A1c, plasma triglycerides, body weight as well as changes inestablished disease bio-markers such as increases in the levels ofadrenomedullin and leptin. Leptin is a known HIF target gene product(Grosfeld et al., 2002, J Biol Chem., 277(45):42953-57). Gene productsinvolved in the metabolism in fat cells were demonstrated to beregulated by PHD inhibition in a HIF-dependent fashion (Intl. Pat. Appl.Publ. WO2004/052285, supra). These include apolipoprotein A-IV, acyl CoAthioesterase, carnitine acetyl transferase, and insulin-like growthfactor binding protein (IGFBP)-1.

PHD inhibitors are expected to be therapeutically useful as stimulantsof vasculogenesis, angiogenesis, and arteriogenesis. These processesestablish or restore blood flow and oxygenation to the tissues underischemia and/or hypoxia conditions (Semenza et al., 2007, J CellBiochem., 102:840-47; Semenza, 2007, Exp Physiol., 92(6):988-91). It hasbeen shown that physical exercise increases HIF-1 and vascularendothelial growth factor in experimental animal models and in humans(Gustafsson et al. 2001, Front Biosci., 6:D75-89) and consequently thenumber of blood vessels in skeletal muscle. VEGF is a well-known HIFtarget gene product that is a key driver of angiogenesis (Liu et al.,supra). While administration of various forms of VEGF receptoractivators are potent stimuli for angiogenesis, the blood vesselresulting from this potential form of therapy are leaky. This isconsidered to limit the potentially utility of VEGF for the treatment ofdisorders of oxygen delivery. The increased expression of a singleangiogenic factor may not be sufficient for functional vascularization(Semenza, 2007, supra). PHD inhibition offers a potential advantage overother such angiogenic therapies in that it stimulates a controlledexpression of multiple angiogenic growth factors in a HIF-dependentfashion including but not limited to: placental growth factor (PLGF),angiopoietin-1 (ANGPT1), angiopoietin-2 (ANGPT2), platelet-derivedgrowth factor beta (PDGFB) (Carmeliet, 2004, J Intern Med., 255:538-61;Kelly et al., 2003, Circ Res., 93:1074-81) and stromal cell derivedfactor 1 (SDF-1) (Ceradini et al., 2004, Nat Med., 10(8):858-64).Expression of angiopoietin-1 during angiogenesis producesleakage-resistant blood vessels, in contrast to the vessels produced byadministration of VEGF alone (Thurston et al., 1999, Science,286:2511-14; Thurston et al., 2000, Nat Med., 6(4):460-3; Elson et al.,2001, Genes Dev., 15(19):2520-32). Stromal cell derived factor 1 (SDF-1)has been shown to be critical to the process of recruiting endothelialprogenitor cells to the sites of tissue injury. SDF-1 expressionincreased the adhesion, migration and homing of circulatingCXCR4-positive progenitor cells to ischemic tissue. Furthermoreinhibition of SDF-1 in ischemic tissue or blockade of CXCR4 oncirculating cells prevents progenitor cell recruitment to sites ofinjury (Ceradini et al., 2004, supra; Ceradini et al., 2005, TrendsCardiovasc Med., 15(2):57-63). Importantly, the recruitment ofendothelial progenitor cells to sites of injury is reduced in aged miceand this is corrected by interventions that increase HIF at the woundsite (Chang et al., 2007, Circulation, 116(24):2818-29). PHD inhibitionoffers the advantage not only of increasing the expression of a numberof angiogenic factions but also a co-ordination in their expressionthroughout the angiogenesis process and recruitment of endothelialprogenitor cells to ischemic tissue.

Evidence for the utility of PHD inhibitors as pro-angiogenic therapiesis provided by the following observations. Adenovirus-mediatedover-expression of HIF has been demonstrated to induce angiogenesis innon-ischemic tissue of an adult animal (Kelly et al., 2003, Circ Res.,93(11):1074-81) providing evidence that therapies that elevate HIF, suchas PHD inhibition, will induce angiogenesis. Placental growth factor(PLGF), also a HIF target gene, has been show to play a critical role inangiogenesis in ischemic tissue (Carmeliet, 2004, J Intern Med.,255(5):538-61; Luttun et al., 2002, Ann N Y Acad Sci., 979:80-93). Thepotent pro-angiogenic effects of therapies that elevate HIF have beendemonstrated, via HIF over-expression, in skeletal muscle (Pajusola etal., 2005, FASEB J., 19(10):1365-7; Vincent et al., 2000, Circulation,102:2255-61) and in the myocardium (Shyu et al., 2002, Cardiovasc Res.,54:576-83). The recruitment of endothelial progenitor cells to theischemic myocardium by the HIF target gene SDF-1 has also beendemonstrated (Abbott et al., 2004, Circulation, 110(21):3300-05). Thesefindings support the general concept that PHD inhibitors will beeffective in stimulating angiogenesis in the setting of tissue ischemia,particularly muscle ischemia. It is expected that therapeuticangiogenesis produced by PHD inhibitors will be useful in restoringblood flow to tissues and therefore the treatment of disease includingbut not restricted to angina pectoris, myocardial ischemia andinfarction, peripheral ischemic disease, claudication, gastric andduodenal ulcers, ulcerative colitis.

PHD and HIF play a central role in tissue repair and regenerationincluding healing of wounds and ulcers. Recent studies have demonstratedthat an increased expression of all three PHDs at wound sites in agedmice with a resulting reduction in HIF accumulation (Chang et al.,supra). Thus, elevation of HIF in aged mice by administeringdesferrioxamine increased the degree of wound healing back to levelsobserved in young mice. Similarly, in a diabetic mouse model, HIFelevation was suppressed compared to non-diabetic litter mates (Mace etal., 2007, Wound Repair Regen., 15(5):636-45). Topical administration ofcobalt chloride, a hypoxia mimetic, or over-expression of a murine HIFthat lacks the oxygen-dependent degradation domain and thus provides fora constitutively active form of HIF, resulted in increased HIF at thewound site, increased expression of HIF target genes such as VEGF, Nos2,and Hmox1 and accelerated wound healing. The beneficial effect of PHDinhibition is not restricted to the skin and small molecule inhibitorsof PHD have recently been demonstrated to provide benefit in a mousemodel of colitis (Robinson et al., 2008, Gastroenterology,134(1):145-55).

PHD inhibition resulting in accumulation of HIF is expected to act by atleast four mechanisms to contribute to accelerated and more completehealing of wounds: 1) protection of tissue jeopardized byhypoxia/ischemia, 2) stimulation of angiogenesis to establish or restoreappropriate blood flow to the site, 3) recruitment of endothelialprogenitor cells to wound sites, 4) stimulation of the release of growthfactors that specifically stimulate healing and regeneration.

Recombinant human platelet-derived growth factor (PDGF) is marketed asbecaplermin (Regranex™) and has been approved by the Food and DrugAdministration of the United States of America for “Treatment of lowerextremity diabetic neuropathic ulcers that extend into the subcutaneoustissue or beyond, and have adequate blood supply”. Becaplermin has beenshown to be effective in accelerating wound healing in diabetic patients(Steed, 2006, Plast Reconstr Surg., 117(7 Suppl):1435-1495; Nagai etal., 2002, Expert Opin Biol Ther., 2(2):211-8). As PDGF is a HIF genetarget (Schultz et al., 2006, Am J Physiol Heart Circ Physiol.,290(6):H2528-34; Yoshida et al., 2006, J Neurooncol., 76(1):13-21), PHDinhibition is expected to increase the expression of endogenous PDGF andproduce a similar or more beneficial effect to those produced withbecaplermin alone. Studies in animals have shown that topicalapplication of PDGF results in increased wound DNA, protein, andhydroxyproline amounts; formation of thicker granulation and epidermaltissue; and increased cellular repopulation of wound sites. PDGF exertsa local effect on enhancing the formation of new connective tissue. Theeffectiveness of PHD inhibition is expected to be greater than thatproduced by becaplermin due to the additional tissue protective andpro-angiogenic effects mediated by HIF.

The beneficial effects of inhibition of PHD are expected to extend notonly to accelerated wound healing in the skin and colon but also to thehealing of other tissue damage including but not limited togastrointestinal ulcers, skin graft replacements, burns, chronic woundsand frost bite.

Stem cells and progenitor cells are found in hypoxic niches within thebody and hypoxia regulates their differentiation and cell fate (Simon etal., 2008, Nat Rev Mol Cell Biol., 9:285-96). Thus PHD inhibitors may beuseful to maintain stem cells and progenitor cells in a pluripotentstate and to drive differentiation to desired cell types. Stem cells maybe useful in culturing and expanding stem cell populations and may holdcells in a pluripotent state while hormones and other factors areadministered to the cells to influence the differentiation and cellfate.

A further use of PHD inhibitors in the area of stem cell and progenitorcell therapeutics relates to the use of PHD inhibitors to conditionthese cells to withstand the process of implantation into the body andto generate an appropriate response to the body to make the stem celland progenitor cell implantation viable (Hu et al., 2008, J ThoracCardiovasc Surg., 135(4):799-808). More specifically PHD inhibitors mayfacilitate the integration of stem cells and draw in an appropriateblood supply to sustain the stem cells once they are integrated. Thisblood vessel formation will also function to carry hormones and otherfactors released from these cells to the rest of the body.

Certain small molecules with Prolyl Hydroxylase antagonistic activitieshave been described in the literature. These include, but are notlimited to, certain imidazo[1,2-a]pyridine derivatives (Warshakoon etal., 2006, Bioorg Med Chem Lett., 16(21):5598-601), substituted pyridinederivatives (Warshakoon et al., 2006, Bioorg Med Chem Lett.,16(21):5616-20), certain pyrazolopyridines (Warshakoon et al., 2006,Bioorg Med Chem Lett., 16(21):5687-90), certain bicyclic heteroaromaticN-substituted glycine derivatives (Intl. Pat. Appl. Publ. WO2007/103905,Sep. 13, 2007), quinoline based compounds (Intl. Pat. Appl. Publ.WO2007/070359, Jun. 21, 2007), certain pyrimidinetrione N-substitutedglycine derivatives (Intl. Pat. Appl. Publ. WO2007/150011, Dec. 27,2007), and substituted aryl or heteroaryl amide compounds (U.S. Pat.Appl. Publ. No.: US 2007/0299086, Dec. 27, 2007). However, there remainsa need for potent prolyl hydroxylase modulators with desirablepharmaceutical properties. Certain benzoimidazole derivatives have beenfound in the context of this invention to have prolyl hydroxylasemodulating activity.

SUMMARY OF THE INVENTION

Certain benzoimidazole derivatives have now been found to have prolylhydroxylase modulating activity. Thus, the invention is directed to thegeneral and preferred embodiments defined, respectively, by theindependent and dependent claims appended hereto, which are incorporatedby reference herein.

In one general aspect the invention relates to a compound of thefollowing Formula (I):

wherein

X is an optionally substituted heteroaryl selected from pyrazole,pyridine, pyrimidine, and pyridazine;

A is a bond or optionally substituted methylene;

each Z is independently Carbon or Nitrogen;

n is 0-4; and

R¹ is independently selected from hydrogen, halogen, nitro, —C₁₋₄alkyl,alkoxy, cycloalkoxy, CON(R^(y))(R^(z)), trifluoromethyl,trifluoromethoxy, N(R^(y))R^(z) (wherein R^(y) and R^(z) areindependently hydrogen, aryl, arylsulfonyl, —C₁₋₆alkyl, and—C₁₋₆alkenyl, or R^(y) and R^(z) may be taken together with the nitrogenof attachment to form an otherwise aliphatic hydrocarbon ring, said ringbeing optionally substituted and having 4 to 7 members, optionallyhaving one carbon replaced with >O, ═N, >NH, or >N(C₁₋₄alkyl), andoptionally substituted Ar wherein said Ar is aryl or heteroaryl.

In another aspect, the invention relates to a compound of the followingFormula (II):

wherein

each Z is independently Carbon or Nitrogen;

n is 0-4;

R¹ is independently selected from hydrogen, halogen, nitro, —C₁₋₄alkyl,alkoxy, cycloalkoxy, CON(R^(y))(R^(z)), trifluoromethyl,trifluoromethoxy, N(R^(y))R^(z) (wherein R^(y) and R^(z) areindependently hydrogen, aryl, arylsulfonyl, —C₁₋₆alkyl, and—C₁₋₆alkenyl, or R^(y) and R^(z) may be taken together with the nitrogenof attachment to form an otherwise aliphatic hydrocarbon ring, said ringbeing optionally substituted and having 4 to 7 members, optionallyhaving one carbon replaced with >O, ═N, >NH, or >N(C₁₋₄alkyl), andoptionally substituted Ar wherein said Ar is aryl or heteroaryl; and

R² is hydrogen, hydroxymethyl or (C₁₋₄)alkyl.

The invention also relates to pharmaceutically acceptable salts,pharmaceutically acceptable prodrugs, and pharmaceutically activemetabolites of compounds of Formula (I) and/or Formula (II). In certainpreferred embodiments, the compound of Formula (I) and/or Formula (II)is a compound selected from those species described or exemplified inthe detailed description below.

In a further general aspect, the invention relates to pharmaceuticalcompositions each comprising: (a) an effective amount of a compound ofFormula (I) and/or Formula (II), or a pharmaceutically acceptable salt,pharmaceutically acceptable prodrug, or pharmaceutically activemetabolite thereof; and (b) a pharmaceutically acceptable excipient.

In another general aspect, the invention is directed to a method oftreating a subject suffering from or diagnosed with a disease, disorder,or medical condition mediated by a prolyl hydroxylase enzyme activity,comprising administering to the subject in need of such treatment aneffective amount of a compound of Formula (I), or a pharmaceuticallyacceptable salt, pharmaceutically acceptable prodrug, orpharmaceutically active metabolite thereof.

In certain preferred embodiments of the inventive method, the disease,disorder, or medical condition is selected from: anemia, vasculardisorders, metabolic disorders, and wound healing.

Additional embodiments, features, and advantages of the invention willbe apparent from the following detailed description and through practiceof the invention.

DETAILED DESCRIPTION

The invention may be more fully appreciated by reference to thefollowing description, including the following glossary of terms and theconcluding examples. For the sake of brevity, the disclosures of thepublications, including patents, cited in this specification are hereinincorporated by reference.

As used herein, the terms “including”, “containing” and “comprising” areused herein in their open, non-limiting sense.

The term “alkyl” refers to a straight- or branched-chain alkyl grouphaving from 1 to 12 carbon atoms in the chain. Examples of alkyl groupsinclude methyl (Me, which also may be structurally depicted by thesymbol, “/”), ethyl (Et), n-propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl (tBu), pentyl, isopentyl, tert-pentyl, hexyl,isohexyl, and groups that in light of the ordinary skill in the art andthe teachings provided herein would be considered equivalent to any oneof the foregoing examples.

The term “cycloalkyl” refers to a saturated or partially saturated,monocyclic, fused polycyclic, or spiro polycyclic carbocycle having from3 to 12 ring atoms per carbocycle. Illustrative examples of cycloalkylgroups include the following entities, in the form of properly bondedmoieties:

A “heterocycloalkyl” refers to a monocyclic ring structure that issaturated or partially saturated and has from 4 to 7 ring atoms per ringstructure selected from carbon atoms and up to two heteroatoms selectedfrom nitrogen, oxygen, and sulfur. The ring structure may optionallycontain up to two oxo groups on sulfur ring members. Illustrativeentities, in the form of properly bonded moieties, include:

The term “heteroaryl” refers to a monocyclic, fused bicyclic, or fusedpolycyclic aromatic heterocycle (ring structure having ring atomsselected from carbon atoms and up to four heteroatoms selected fromnitrogen, oxygen, and sulfur) having from 3 to 12 ring atoms perheterocycle. Illustrative examples of heteroaryl groups include thefollowing entities, in the form of properly bonded moieties:

Those skilled in the art will recognize that the species of cycloalkyl,heterocycloalkyl, and heteroaryl groups listed or illustrated above arenot exhaustive, and that additional species within the scope of thesedefined terms may also be selected.

The term “halogen” represents chlorine, fluorine, bromine or iodine. Theterm “halo” represents chloro, fluoro, bromo or iodo.

The term “substituted” means that the specified group or moiety bearsone or more substituents. The term “unsubstituted” means that thespecified group bears no substituents. The term “optionally substituted”means that the specified group is unsubstituted or substituted by one ormore substituents. Where the term “substituted” is used to describe astructural system, the substitution is meant to occur at anyvalency-allowed position on the system. In cases where a specifiedmoiety or group is not expressly noted as being optionally substitutedor substituted with any specified substituent, it is understood thatsuch a moiety or group is intended to be unsubstituted.

Any formula given herein is intended to represent compounds havingstructures depicted by the structural formula as well as certainvariations or forms. In particular, compounds of any formula givenherein may have asymmetric centers and therefore exist in differentenantiomeric forms. All optical isomers and stereoisomers of thecompounds of the general formula, and mixtures thereof, are consideredwithin the scope of the formula. Thus, any formula given herein isintended to represent a racemate, one or more enantiomeric forms, one ormore diastereomeric forms, one or more atropisomeric forms, and mixturesthereof. Furthermore, certain structures may exist as geometric isomers(i.e., cis and trans isomers), as tautomers, or as atropisomers.Additionally, any formula given herein is intended to embrace hydrates,solvates, and polymorphs of such compounds, and mixtures thereof.

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds. Isotopicallylabeled compounds have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number. Examples of isotopes that can beincorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine,such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl,¹²⁵I, respectively. Such isotopically labeled compounds are useful inmetabolic studies (preferably with ¹⁴C), reaction kinetic studies (with,for example ²H or ³H), detection or imaging techniques [such as positronemission tomography (PET) or single-photon emission computed tomography(SPECT)] including drug or substrate tissue distribution assays, or inradioactive treatment of patients. In particular, an ¹⁸F or ¹¹C labeledcompound may be particularly preferred for PET or SPECT studies.Further, substitution with heavier isotopes such as deuterium (i.e., ²H)may afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements. Isotopically labeled compounds of this inventionand prodrugs thereof can generally be prepared by carrying out theprocedures disclosed in the schemes or in the examples and preparationsdescribed below by substituting a readily available isotopically labeledreagent for a non-isotopically labeled reagent.

When referring to any formula given herein, the selection of aparticular moiety from a list of possible species for a specifiedvariable is not intended to define the moiety for the variable appearingelsewhere. In other words, where a variable appears more than once, thechoice of the species from a specified list is independent of the choiceof the species for the same variable elsewhere in the formula.

Chemical depictions are intended to portray the compound portionscontaining the orientations as written.

The instant invention includes the use of compounds of Formula (I)and/or Formula (II) and pharmaceutical compositions containing suchcompounds thereof to treat patients (humans or other mammals) withdisorders related to the modulation of the prolyl hydroxylase enzyme.The instant invention also includes methods of making such a compound,pharmaceutical composition, pharmaceutically acceptable salt,pharmaceutically acceptable prodrug, and pharmaceutically activemetabolites thereof.

In preferred embodiments of Formula (I), X is selected from the groupconsisting of an optionally substituted pyrazole, pyridine, pyrimidine,and pyridazine. In a related embodiment, X is an unsubstituted pyrazole.In an additional embodiment, X can be an optionally substitutedpyridine, pyrimidine, or pyridazine wherein said pyridine, pyrimidine,or pyridazine is substituted with one or more substituents independentlyselected from hydrogen, halo, hydroxyl, methyl or CF₃.

In one embodiment, A is a bond or methlyene optionally substituted withhydrogen, hydroxymethyl or —C₁₋₄alkyl. In further preferred embodiment,A is a bond.

In preferred embodiments for Formula (I) and/or Formula (II), each R¹ isindependently selected from hydrogen, halogen, nitro, —C₁₋₄alkyl,alkoxy, cycloalkoxy, CON(R^(y))(R^(z)), trifluoromethyl,trifluoromethoxy, N(R^(y))R^(z) (wherein R^(y) and R^(z) areindependently hydrogen, aryl, arylsulfonyl, —C₁₋₆alkyl, and—C₁₋₆alkenyl, or R^(y) and R^(z) may be taken together with the nitrogenof attachment to form an otherwise aliphatic hydrocarbon ring, said ringbeing optionally substituted and having 4 to 7 members, optionallyhaving one carbon replaced with >O, ═N, >NH, or >N(C₁₋₄alkyl), andoptionally substituted Ar wherein said Ar is aryl or heteroaryl.

In preferred embodiments of Formula (I) and/or Formula (II), each R¹ isindependently selected from the group consisting of hydrogen,—C₁₋₄alkyl, halo, —C₁₋₄alkoxy, —NO₂, 2-hydroxy-phenyl, 3-hydroxy-phenyl,4-hydroxy-phenyl, trifluoromethyl, trifluoromethoxy, 3-hydroxyphenyl,4-hydroxyphenyl, 3-benzyloxy-phenyl, 3-(2-chloro-benzyloxy)-phenyl,3-(3-chloro-benzyloxy)-phenyl, 3-(4-chloro-benzyloxy)-phenyl,quinolin-3-yl, 4-(2-fluoro-benzyloxy)-phenyl,4-(3-fluoro-benzyloxy)-phenyl, 4-(4-fluoro-benzyloxy)-phenyl,4-(3-chloro-benzyloxy)-phenyl, 4-benzyloxy-3-fluoro-phenyl,4-benzyloxy-2-fluoro-phenyl, 4-phenoxy-phenyl, benzyl-amine,2-naphthalen-2-yl-phenyl, 3-naphthalen-2-yl-phenyl,4-naphthalen-2-yl-phenyl, 4-(2-chloro-benzyloxy)-3,5-dimethyl-phenyl,4-(3-chloro-benzyloxy)-3,5-dimethyl-phenyl,4-(4-chloro-benzyloxy)-3,5-dimethyl-phenyl, 4-propoxy-phenyl,5-chloro-2-fluoro-phenyl, benzenesulfonamide, 2-methoxy-phenyl,3-methoxy-phenyl, 4-methoxy-phenyl, or 4-chloro-3-methyl-phenyl.

In another embodiment of Formula (I) and/or Formula (II), individual R¹comprises an electron withdrawing group. In a further embodiment, saidelectron withdrawing

group is in the X position according to the formula

In preferred embodiments of Formula (II), R² is hydrogen, hydroxymethyl,or —C₁₋₄alkyl.

In certain preferred embodiments, the compound of Formula (I) or Formula(II) is selected from the group consisting of:

Cellular % Enzyme EPO Ex. Chemical Name pIC50 Stimulation 1[(1H-Benzoimidazole-2-carbonyl)-amino]-acetic acid <5 23.9 2(S)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-propionic 4.40 66.03 acid 3[(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)-amino]- 4.90 8.77 aceticacid 4 (S)-2-[(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)- 4.60 7.70amino]-propionic acid 5[(6-Nitro-1H-benzoimidazole-2-carbonyl)-amino]-acetic 5.30 0.00 acid 6[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2-carbonyl)- 4.70 4.20amino]-acetic acid 7(S)-2-[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2- 4.30 1.60carbonyl)-amino]-propionic acid 8[(5-Iodo-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid 4.40 3.80 9[(6-Bromo-1H-benzoimidazole-2-carbonyl)-amino]-acetic 4.60 9.80 acid 10(S)-2-[(6-Fluoro-1H-benzoimidazole-2-carbonyl)-amino]- <4 14.74propionic acid 11 (R)-2-[(6-Fluoro-1H-benzoimidazole-2-carbonyl)-amino]-4.10 23.93 propionic acid 12[(6-Methoxy-1H-benzoimidazole-2-carbonyl)-amino]-acetic 4.30 0.50 acid13 ({5-[3-(3-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2- 5.80 1.20carbonyl}-amino)-acetic acid 14({5-[3-(2-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2- 5.60 −3.80carbonyl}-amino)-acetic acid 15({5-[3-(4-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2- 5.60 −3.90carbonyl}-amino)-acetic acid 16({5-[3-(3-Fluoro-benzyloxy)-phenyl]-1H-benzoimidazole-2- 5.60 2.10carbonyl}-amino)-acetic acid 17({5-[3-(2-Fluoro-benzyloxy)-phenyl]-1H-benzoimidazole-2- 5.30 1.50carbonyl}-amino)-acetic acid 18{[5-(4-Phenoxy-phenyl)-1H-benzoimidazole-2-carbonyl]- 5.10 2.00amino}-acetic acid 19{[5-(3-Benzyloxy-5-fluoro-phenyl)-1H-benzoimidazole-2- 5.20 −3.80carbonyl]-amino}-acetic acid 20[(5-Quinolin-3-yl-1H-benzoimidazole-2-carbonyl)-amino]- 5.80 8.26 aceticacid 21 ({5-[3-Chloro-4-(3-chloro-benzyloxy)phenyl]-1H- 5.10 −1.43benzoimidazole-2-carbonyl}-amino)-acetic acid 22({5-[4-(3-Chloro-benzyloxy)-3,5-dimethyl-phenyl]-1H- 4.90 −1.06benzoimidazole-2-carbonyl}-amino)-acetic acid 23{[5-(3-Methoxy-phenyl)-1H-benzoimidazole-2-carbonyl]- 4.40 1.40amino}-acetic acid 24 {[5-(5-Benzylcarbamoyl-2-fluoro-phenyl)-1H- 4.8013.56 benzoimidazole-2-carbonyl]-amino}-acetic acid 25[(5-Naphthalen-2-yl-1H-benzoimidazole-2-carbonyl)- 4.90 2.10amino]-acetic acid 26{[5-(4-Propoxy-phenyl)-1H-benzoimidazole-2-carbonyl]- 4.80 2.50amino}-acetic acid 27{[5-(4-Chloro-3-methyl-phenyl)-1H-benzoimidazole-2- 4.40 −0.70carbonyl]-amino}-acetic acid 28{[5-(5-Chloro-2-fluoro-phenyl)-1H-benzoimidazole-2- 4.60 1.40carbonyl]-amino}-acetic acid 29[(5-Benzoylamino-1H-benzoimidazole-2-carbonyl)-amino]- 4.20 −3.30 aceticacid 30 [(6-Benzenesulfonylamino-1H-benzoimidazole-2- 4.50 1.10carbonyl)-amino]-acetic acid 31[(6-Benzylamino-1H-benzoimidazole-2-carbonyl)-amino]- 5.30 1.40 aceticacid 32 1-(1H-Benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid 6.107.10 33 1-(5,6-Dichloro-1H-benzoimidazol-2-yl)-1H-pyrazole-4- 7.00168.46 carboxylic acid 341-(5-Trifluoromethyl-1H-benzoimidazol-2-yl)-1H-pyrazole- 6.60 127.804-carboxylic acid 351-(5-Chloro-6-fluoro-1H-benzoimidazol-2-yl)-1H-pyrazole- 6.70 104.604-carboxylic acid 361-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4- 6.10 2.10carboxylic acid 37 1-(5-Bromo-1H-benzoimidazol-2-yl)-1H-pyrazole-4- 6.409.91 carboxylic acid 381-(5-Methoxy-1H-benzoimidazol-2-yl)-1H-pyrazole-4- 6.40 16.80 carboxylicacid 39 1-(4-Chloro-6-trifluoromethyl-1H-benzoimidazol-2-yl)-1H- 6.8020.97 pyrazole-4-carboxylic acid 401-(5,6-Dimethoxy-1H-benzoimidazol-2-yl)-1H-pyrazole-4- 6.40 4.26carboxylic acid 41 1-(4,5-Dimethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-5.10 10.70 carboxylic acid 421-(5-Trifluoromethoxy-1H-benzoimidazol-2-yl)-1H- 6.50 123.68pyrazole-4-carboxylic acid 431-{5-[3-(3-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2- 7.50 8.76yl}-1H-pyrazole-4-carboxylic acid 441-{5-[3-(2-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2- 7.60 18.00yl}-1H-pyrazole-4-carboxylic acid 451-{5-[3-(4-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2- 7.40 2.10yl}-1H-pyrazole-4-carboxylic acid 461-[5-(3-Benzyloxy-phenyl)-1H-benzoimidazol-2-yl]-1H- 7.30 10.77pyrazole-4-carboxylic acid 471-[5-(4-Benzyloxy-phenyl)-1H-benzoimidazol-2-yl]-1H- 6.70 6.97pyrazole-4-carboxylic acid 481-[5-(3-Trifluoromethyl-phenyl)-1H-benzoimidazol-2-yl]- 7.10 5.801H-pyrazole-4-carboxylic acid 491-[5-(3,4-Dichloro-phenyl)-1H-benzoimidazol-2-yl]-1H- 7.10 15.30pyrazole-4-carboxylic acid 50(R)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-propionic <4 9.50* acid 51(R)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-3-hydroxy- 4.70 0.73*propionic acid 52 (R)-2-[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2-4.00 17.80 carbonyl)-amino]-propionic acid 53(R)-2-[(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)- <4 11.3*amino]-propionic acid 54(R)-2-[(5-Iodo-1H-benzoimidazole-2-carbonyl)-amino]- <4 7.50 propionicacid 55 1-(5-Bromo-1H-benzoimidazol-2-yl)-3-trifluoromethyl-1H- <4 20.52pyrazole-4-carboxylic acid 561-(5,6-dichloro-1H-benzoimidazol-2-yl)-3-trifluoromethyl- <5 18.811H-pyrazole-4-carboxylic acid 571-(5-Bromo-1H-benzoimidazol-2-yl)-3,5-dimethyl-1H- 4.20 7.24pyrazole-4-carboxylic acid 581-(5,6-Dichloro-1H-benzoimidazol-2-yl)-3,5-dimethyl-1H- <5 0.47pyrazole-4-carboxylic acid 591-[5-(4-Hydroxy-phenyl)-1H-benzoimidazol-2-yl]-1H- 6.60 6.49pyrazole-4-carboxylic acid 601-[5-(3-Hydroxy-phenyl)-1H-benzoimidazol-2-yl]-1H- 6.50 11.15pyrazole-4-carboxylic acid 611-(5-Chloro-1H-benzoimidazol-2-yl)-1H-pyrazole-4- 6.30 71.87 carboxylicacid 62 1-(5-Bromo-6,7-dimethyl-1H-benzoimidazol-2-yl)-1H- 6.40 11.90pyrazole-4-carboxylic acid *final compound concentration of 10 μM andpharmaceutically acceptable salts thereof.

The invention includes also pharmaceutically acceptable salts of thecompounds of Formula (I), preferably of those described above and of thespecific compounds exemplified herein, and methods of treatment usingsuch salts.

A “pharmaceutically acceptable salt” is intended to mean a salt of afree acid or base of a compound represented by Formula (I) that isnon-toxic, biologically tolerable, or otherwise biologically suitablefor administration to the subject. See, generally, S. M. Berge, et al.,“Pharmaceutical Salts”, J Pharm Sci., 1977, 66:1-19, and Handbook ofPharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth,Eds., Wiley-VCH and VHCA, Zurich, 2002. Examples of pharmaceuticallyacceptable salts are those that are pharmacologically effective andsuitable for contact with the tissues of patients without unduetoxicity, irritation, or allergic response. A compound of Formula (I)may possess a sufficiently acidic group, a sufficiently basic group, orboth types of functional groups, and accordingly react with a number ofinorganic or organic bases, and inorganic and organic acids, to form apharmaceutically acceptable salt. Examples of pharmaceuticallyacceptable salts include sulfates, pyrosulfates, bisulfates, sulfites,bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates,metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates,propionates, decanoates, caprylates, acrylates, formates, isobutyrates,caproates, heptanoates, propiolates, oxalates, malonates, succinates,suberates, sebacates, fumarates, maleates, butyne-1,4-dioates,hexyne-1,6-dioates, benzoates, chlorobenzoates, methyl benzoates,dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates,sulfonates, xylenesulfonates, phenylacetates, phenylpropionates,phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates,tartrates, methane-sulfonates, propanesulfonates,naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.

If the compound of Formula (I) contains a basic nitrogen, the desiredpharmaceutically acceptable salt may be prepared by any suitable methodavailable in the art, for example, treatment of the free base with aninorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuricacid, sulfamic acid, nitric acid, boric acid, phosphoric acid, and thelike, or with an organic acid, such as acetic acid, phenylacetic acid,propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid,hydroxymaleic acid, isethionic acid, succinic acid, valeric acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, oleic acid, palmitic acid, lauric acid, a pyranosidylacid, such as glucuronic acid or galacturonic acid, an alpha-hydroxyacid, such as mandelic acid, citric acid, or tartaric acid, an aminoacid, such as aspartic acid or glutamic acid, an aromatic acid, such asbenzoic acid, 2-acetoxybenzoic acid, naphthoic acid, or cinnamic acid, asulfonic acid, such as laurylsulfonic acid, p-toluenesulfonic acid,methanesulfonic acid, ethanesulfonic acid, any compatible mixture ofacids such as those given as examples herein, and any other acid andmixture thereof that are regarded as equivalents or acceptablesubstitutes in light of the ordinary level of skill in this technology.

If the compound of Formula (I) is an acid, such as a carboxylic acid orsulfonic acid, the desired pharmaceutically acceptable salt may beprepared by any suitable method, for example, treatment of the free acidwith an inorganic or organic base, such as an amine (primary, secondaryor tertiary), an alkali metal hydroxide, alkaline earth metal hydroxide,any compatible mixture of bases such as those given as examples herein,and any other base and mixture thereof that are regarded as equivalentsor acceptable substitutes in light of the ordinary level of skill inthis technology. Illustrative examples of suitable salts include organicsalts derived from amino acids, such as glycine and arginine, ammonia,carbonates, bicarbonates, primary, secondary, and tertiary amines, andcyclic amines, such as benzylamines, pyrrolidines, piperidine,morpholine, and piperazine, and inorganic salts derived from sodium,calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum,and lithium.

The invention also relates to pharmaceutically acceptable prodrugs ofthe compounds of Formula (I), and treatment methods employing suchpharmaceutically acceptable prodrugs. The term “prodrug” means aprecursor of a designated compound that, following administration to asubject, yields the compound in vivo via a chemical or physiologicalprocess such as solvolysis or enzymatic cleavage, or under physiologicalconditions (e.g., a prodrug on being brought to physiological pH isconverted to the compound of Formula (I)). A “pharmaceuticallyacceptable prodrug” is a prodrug that is non-toxic, biologicallytolerable, and otherwise biologically suitable for administration to thesubject. Illustrative procedures for the selection and preparation ofsuitable prodrug derivatives are described, for example, in “Design ofProdrugs”, ed. H. Bundgaard, Elsevier, 1985.

Exemplary prodrugs include compounds having an amino acid residue, or apolypeptide chain of two or more (e.g., two, three or four) amino acidresidues, covalently joined through an amide or ester bond to a freeamino, hydroxy, or carboxylic acid group of a compound of Formula (I).Examples of amino acid residues include the twenty naturally occurringamino acids, commonly designated by three letter symbols, as well as4-hydroxyproline, hydroxylysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline homocysteine, homoserine, ornithine and methionine sulfone.

Additional types of prodrugs may be produced, for instance, byderivatizing free carboxyl groups of structures of Formula (I) as amidesor alkyl esters. Examples of amides include those derived from ammonia,primary C₁₋₆alkyl amines and secondary di(C₁₋₆alkyl) amines. Secondaryamines include 5- or 6-membered heterocycloalkyl or heteroaryl ringmoieties. Examples of amides include those that are derived fromammonia, C₁₋₃alkyl primary amines, and di(C₁₋₂alkyl)amines. Examples ofesters of the invention include C₁₋₇alkyl, C₅₋₇cycloalkyl, phenyl, andphenyl(C₁₋₆alkyl) esters. Preferred esters include methyl esters.Prodrugs may also be prepared by derivatizing free hydroxy groups usinggroups including hemisuccinates, phosphate esters,dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, followingprocedures such as those outlined in Adv. Drug Delivery Rev. 1996, 19,115. Carbamate derivatives of hydroxy and amino groups may also yieldprodrugs. Carbonate derivatives, sulfonate esters, and sulfate esters ofhydroxy groups may also provide prodrugs. Derivatization of hydroxygroups as (acyloxy)methyl and (acyloxy)ethyl ethers, wherein the acylgroup may be an alkyl ester, optionally substituted with one or moreether, amine, or carboxylic acid functionalities, or where the acylgroup is an amino acid ester as described above, is also useful to yieldprodrugs. Prodrugs of this type may be prepared as described in J MedChem. 1996, 39, 10. Free amines can also be derivatized as amides,sulfonamides or phosphonamides. All of these prodrug moieties mayincorporate groups including ether, amine, and carboxylic acidfunctionalities.

The present invention also relates to pharmaceutically activemetabolites of the compounds of Formula (I), which may also be used inthe methods of the invention. A “pharmaceutically active metabolite”means a pharmacologically active product of metabolism in the body of acompound of Formula (I) or salt thereof. Prodrugs and active metabolitesof a compound may be determined using routine techniques known oravailable in the art. See, e.g., Bertolini, et al., J Med Chem. 1997,40, 2011-2016; Shan, et al., J Pharm Sci. 1997, 86 (7), 765-767;Bagshawe, Drug Dev Res. 1995, 34, 220-230; Bodor, Adv Drug Res. 1984,13, 224-331; Bundgaard, Design of Prodrugs (Elsevier Press, 1985); andLarsen, Design and Application of Prodrugs, Drug Design and Development(Krogsgaard-Larsen, et al., eds., Harwood Academic Publishers, 1991).

The compounds of Formula (I) and their pharmaceutically acceptablesalts, pharmaceutically acceptable prodrugs, and pharmaceutically activemetabolites of the present invention are useful as modulators of PHD inthe methods of the invention. As such modulators, the compounds may actas antagonists, agonists, or inverse agonists. “Modulators” include bothinhibitors and activators, where “inhibitors” refer to compounds thatdecrease, prevent, inactivate, desensitize or down-regulate PHDexpression or activity, and “activators” are compounds that increase,activate, facilitate, sensitize, or up-regulate PHD expression oractivity.

The term “treat” or “treating” as used herein is intended to refer toadministration of an active agent or composition of the invention to asubject for the purpose of effecting a therapeutic or prophylacticbenefit through modulation of prolyl hydroxylase activity. Treatingincludes reversing, ameliorating, alleviating, inhibiting the progressof, lessening the severity of, or preventing a disease, disorder, orcondition, or one or more symptoms of such disease, disorder orcondition mediated through modulation of PHD activity. The term“subject” refers to a mammalian patient in need of such treatment, suchas a human.

Accordingly, the invention relates to methods of using the compoundsdescribed herein to treat subjects diagnosed with or suffering from adisease, disorder, or condition mediated by Prolyl Hydroxylase, such as:Anemia, vascular disorders, metabolic disorders, and wound healing.Symptoms or disease states are intended to be included within the scopeof “medical conditions, disorders, or diseases.”

In a preferred embodiment, molecules of the present invention are usefulin the treatment or prevention of anemia comprising treatment of anemicconditions associated with chronic kidney disease, polycystic kidneydisease, aplastic anemia, autoimmune hemolytic anemia, bone marrowtransplantation anemia, Churg-Strauss syndrome, Diamond Blackfan anemia,Fanconi's anemia, Felty syndrome, graft versus host disease,hematopoietic stem cell transplantation, hemolytic uremic syndrome,myelodysplastic syndrome, nocturnal paroxysmal hemoglobinuria,osteomyelofibrosis, pancytopenia, pure red-cell aplasia, purpuraSchoenlein-Henoch, refractory anemia with excess of blasts, rheumatoidarthritis, Shwachman syndrome, sickle cell disease, thalassemia major,thalassemia minor, thrombocytopenic purpura, anemic or non-anemicpatients undergoing surgery, anemia associated with or secondary totrauma, sideroblastic anemia, anemic secondary to other treatmentincluding: reverse transcriptase inhibitors to treat HIV, corticosteroidhormones, cyclic cisplatin or non-cisplatin-containingchemotherapeutics, vinca alkaloids, mitotic inhibitors, topoisomerase IIinhibitors, anthracyclines, alkylating agents, particularly anemiasecondary to inflammatory, aging and/or chronic diseases. PHD inhibitionmay also be used to treat symptoms of anemia including chronic fatigue,pallor and dizziness.

In another preferred embodiment, molecules of the present invention areuseful in the treatment or prevention of diseases of metabolicdisorders, including but not limited to diabetes and obesity. In anotherpreferred embodiment, molecules of the present invention are useful inthe treatment or prevention of vascular disorders. These include but arenot limited to hypoxic or wound healing related diseases requiringpro-angiogenic mediators for vasculogenesis, angiogenesis, andarteriogenesis

“Modulators” include both inhibitors and activators, where “inhibitors”refer to compounds that decrease, prevent, inactivate, desensitize ordown-regulate PHD expression or activity, and “activators” are compoundsthat increase, activate, facilitate, sensitize, or up-regulate PHDexpression or activity.

In treatment methods according to the invention, an effective amount ofa pharmaceutical agent according to the invention is administered to asubject suffering from or diagnosed as having such a disease, disorder,or condition. An “effective amount” means an amount or dose sufficientto generally bring about the desired therapeutic or prophylactic benefitin patients in need of such treatment for the designated disease,disorder, or condition. Effective amounts or doses of the compounds ofthe present invention may be ascertained by routine methods such asmodeling, dose escalation studies or clinical trials, and by taking intoconsideration routine factors, e.g., the mode or route of administrationor drug delivery, the pharmacokinetics of the compound, the severity andcourse of the disease, disorder, or condition, the subject's previous orongoing therapy, the subject's health status and response to drugs, andthe judgment of the treating physician. An example of a dose is in therange of from about 0.001 to about 200 mg of compound per kg ofsubject's body weight per day, preferably about 0.05 to 100 mg/kg/day,or about 1 to 35 mg/kg/day, in single or divided dosage units (e.g.,BID, TID, QID). For a 70-kg human, an illustrative range for a suitabledosage amount is from about 0.05 to about 7 g/day, or about 0.2 to about2.5 g/day.

Once improvement of the patient's disease, disorder, or condition hasoccurred, the dose may be adjusted for preventative or maintenancetreatment. For example, the dosage or the frequency of administration,or both, may be reduced as a function of the symptoms, to a level atwhich the desired therapeutic or prophylactic effect is maintained. Ofcourse, if symptoms have been alleviated to an appropriate level,treatment may cease. Patients may, however, require intermittenttreatment on a long-term basis upon any recurrence of symptoms.

In addition, the agents of the invention may be used in combination withadditional active ingredients in the treatment of the above conditions.The additional compounds may be co-administered separately with an agentof Formula (I) or included with such an agent as an additional activeingredient in a pharmaceutical composition according to the invention.In an exemplary embodiment, additional active ingredients are those thatare known or discovered to be effective in the treatment of conditions,disorders, or diseases mediated by PHD enzyme or that are active againstanother targets associated with the particular condition, disorder, ordisease, such as an alternate PHD modulator. The combination may serveto increase efficacy (e.g., by including in the combination a compoundpotentiating the potency or effectiveness of a compound according to theinvention), decrease one or more side effects, or decrease the requireddose of the compound according to the invention.

The compounds of the invention are used, alone or in combination withone or more other active ingredients, to formulate pharmaceuticalcompositions of the invention. A pharmaceutical composition of theinvention comprises: (a) an effective amount of a compound of Formula(I), or a pharmaceutically acceptable salt, pharmaceutically acceptableprodrug, or pharmaceutically active metabolite thereof; and (b) apharmaceutically acceptable excipient.

A “pharmaceutically acceptable excipient” refers to a substance that isnon-toxic, biologically tolerable, and otherwise biologically suitablefor administration to a subject, such as an inert substance, added to apharmacological composition or otherwise used as a vehicle, carrier, ordiluent to facilitate administration of a compound of the invention andthat is compatible therewith. Examples of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils, and polyethyleneglycols.

Delivery forms of the pharmaceutical compositions containing one or moredosage units of the compounds of the invention may be prepared usingsuitable pharmaceutical excipients and compounding techniques now orlater known or available to those skilled in the art. The compositionsmay be administered in the inventive methods by oral, parenteral,rectal, topical, or ocular routes, or by inhalation. The preparation maybe in the form of tablets, capsules, sachets, dragees, powders,granules, lozenges, powders for reconstitution, liquid preparations, orsuppositories. Preferably, the compositions are formulated forintravenous infusion, topical administration, or oral administration. Apreferred mode of use of the invention is local administration of PHDinhibitors particularly to sites where tissue has become or has beenmade ischemic. This may be achieved via a specialized catheter,angioplasty balloon or stent placement balloon.

For oral administration, the compounds of the invention can be providedin the form of tablets or capsules, or as a solution, emulsion, orsuspension. To prepare the oral compositions, the compounds may beformulated to yield a dosage of, e.g., from about 0.05 to about 100mg/kg daily, or from about 0.05 to about 35 mg/kg daily, or from about0.1 to about 10 mg/kg daily.

Oral tablets may include a compound according to the invention mixedwith pharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservative agents.Suitable inert fillers include sodium and calcium carbonate, sodium andcalcium phosphate, lactose, starch, sugar, glucose, methyl cellulose,magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquidoral excipients include ethanol, glycerol, water, and the like. Starch,polyvinyl-pyrrolidone (PVP), sodium starch glycolate, microcrystallinecellulose, and alginic acid are suitable disintegrating agents. Bindingagents may include starch and gelatin. The lubricating agent, ifpresent, may be magnesium stearate, stearic acid or talc. If desired,the tablets may be coated with a material such as glyceryl monostearateor glyceryl distearate to delay absorption in the gastrointestinaltract, or may be coated with an enteric coating.

Capsules for oral administration include hard and soft gelatin capsules.To prepare hard gelatin capsules, compounds of the invention may bemixed with a solid, semi-solid, or liquid diluent. Soft gelatin capsulesmay be prepared by mixing the compound of the invention with water, anoil such as peanut oil or olive oil, liquid paraffin, a mixture of monoand di-glycerides of short chain fatty acids, polyethylene glycol 400,or propylene glycol.

Liquids for oral administration may be in the form of suspensions,solutions, emulsions or syrups or may be presented as a dry product forreconstitution with water or other suitable vehicle before use. Suchliquid compositions may optionally contain: pharmaceutically-acceptableexcipients such as suspending agents (for example, sorbitol, methylcellulose, sodium alginate, gelatin, hydroxyethylcellulose,carboxymethylcellulose, aluminum stearate gel and the like); non-aqueousvehicles, e.g., oil (for example, almond oil or fractionated coconutoil), propylene glycol, ethyl alcohol, or water; preservatives (forexample, methyl or propyl p-hydroxybenzoate or sorbic acid); wettingagents such as lecithin; and, if desired, flavoring or coloring agents.

The active agents of this invention may also be administered by non-oralroutes. For example, the compositions may be formulated for rectaladministration as a suppository. For parenteral use, includingintravenous, intramuscular, intraperitoneal, or subcutaneous routes, thecompounds of the invention may be provided in sterile aqueous solutionsor suspensions, buffered to an appropriate pH and isotonicity or inparenterally acceptable oil. Suitable aqueous vehicles include Ringer'ssolution and isotonic sodium chloride. Such forms will be presented inunit-dose form such as ampules or disposable injection devices, inmulti-dose forms such as vials from which the appropriate dose may bewithdrawn, or in a solid form or pre-concentrate that can be used toprepare an injectable formulation. Illustrative infusion doses may rangefrom about 1 to 1000 μg/kg/minute of compound, admixed with apharmaceutical carrier over a period ranging from several minutes toseveral days.

For topical administration, the compounds may be mixed with apharmaceutical carrier at a concentration of about 0.1% to about 10% ofdrug to vehicle. Another mode of administering the compounds of theinvention may utilize a patch formulation to affect transdermaldelivery.

Compounds of the invention may alternatively be administered in methodsof this invention by inhalation, via the nasal or oral routes, e.g., ina spray formulation also containing a suitable carrier.

Exemplary compounds useful in methods of the invention will now bedescribed by reference to the illustrative synthetic schemes for theirgeneral preparation below and the specific examples that follow.Artisans will recognize that, to obtain the various compounds herein,starting materials may be suitably selected so that the ultimatelydesired substituents will be carried through the reaction scheme with orwithout protection as appropriate to yield the desired product.Alternatively, it may be necessary or desirable to employ, in the placeof the ultimately desired substituent, a suitable group that may becarried through the reaction scheme and replaced as appropriate with thedesired substituent. Unless otherwise specified, the variables are asdefined above in reference to Formula (I). Reactions may be performedbetween the melting point and the reflux temperature of the solvent, andpreferably between 0° C. and the reflux temperature of the solvent.

Referring to Scheme A, imidazoles A1 are coupled with appropriate aminesusing a peptide coupling reagent such as HATU in the presence of a basesuch as DIPEA to provide A2. When Z═CO₂R, exposure to aqueous base oracid provides A3.

An alternative method for preparing A2 is shown in Scheme B. Brominatedimidazoles B1 (prepared as described in Scheme A) are protected with asuitable reagent such as di-tert-butyl-dicarbonate to provide B2.Coupling reaction with boronic acids or similar organic nucleophile inthe presence of a organotransition metal catalyst such as PdCl₂(dppf)and a base such as CsF affords B3. Removal of the protective group iseffected by exposure to an acid such as trifluoroacetic acid to give A2.

A method to obtain certain benzoimidazoles A1 that are not commerciallyavailable or previously known is shown in Scheme C. Known imidazole C1is coupled to appropriate acid chlorides or sulfonyl chlorides in thepresence of a base such as DIPEA to give C2. Hydrolysis is achievedusing a base such as LiOH in THF/H₂O, or aqueous acid providing A1.

An alternative method to obtain C2 is shown in Scheme D. Subjecting C1to reductive amination conditions with appropriate aldehydes in thepresence of a reducing agent such as sodium triacetoxyborohydrideprovides C2.

Referring to Scheme E, imidazoles E1 are protected using a suitablereagent such as SEMCl or MEMCl in the presence of a base such as NaH orDIPEA to afford E2. Heating E2 with pyrazoles in the presence of a basesuch as Cs₂CO₃ provides E3. Deprotection occurs under acidic conditionsto give E4. Hydrolysis when B═CO₂R is effected with a base such as LiOHin THF/H₂O or aqueous acid to deliver E5.

An alternative preparation of E3 is shown is Scheme F. Brominatedimidazoles F1 (prepared as described in Scheme E) are subjected to anorganotransition metal mediated coupling reaction, such as a Suzukireaction, with appropriate organic nucleophile, such as a boronic acid,in the presence of a catalyst such as PdCl₂(dppf) and a base such as CsFto supply E3.

Referring to Scheme G, in one embodiment, protected2-bromobenzoimidazoles G1 can be treated with organic nucleophiles suchas boronic acids or esters G2 under organotransition metal couplingreaction conditions such as the Suzuki reaction to provide G3. Whenprotecting groups such as MEM or SEM are used, deprotection can beeffected with acid to provide G4. Hydrolysis when Y═CO₂R can be effectedwith a base such as LiOH in THF/H₂O or aqueous acid to provide G5. As analternative method, G6 can be coupled with 2-halopyridine or2-halopyrimidine or 2-halopyridazine derivatives such as G7 under Suzukireaction conditions to provide G3. In yet another alternative method, G4can be accessed by coupling G8 with G9 under oxidative cyclizationconditions.

Referring to Scheme H, in one embodiment H1 can be reacted with areagent such as CU in THF to provide the cyclic urea H2. Treatment of H2with a chlorinating agent such as POCl₃ at moderate to high temperaturescan then provide H3. Protection of imidazoles H3 can be achieved using asuitable reagent such as SEMCl or MEMCl in the presence of a base suchas NaH or DIPEA to provide H4. In one embodiment, heating H4 withsubstituted pyrazoles in the presence of a base such as Cs₂CO₃ can thenprovide H5. Deprotection can be carried out under acidic conditions toyield H6. Hydrolysis when A or B=CO₂R can be effected with a base suchas LiOH in THF/H₂O or aqueous acid to provide H7. In another embodimentH3 can be coupled with pyrazoles to provide H6 without use of protectinggroups.

Referring to Scheme I, hydrolysis of ester I1 can be carried out with abase such as LiOH in THF/H₂O or aqueous acid to provide I2. In oneembodiment, I2 can be homologated using well-known procedures such asthe Arndt-Eistert reaction to provide I3. Deprotection under acidicconditions can then provide I4.

Referring to Scheme J, in one embodiment J1 can be coupled with organicnucleophiles such as boronic acids or esters J2 under organotransitionmetal catalyzed coupling reactions such as the Suzuki reaction toprovide J3. Deprotection under acidic conditions can then provide J4.Hydrolysis of the ester moiety can be effected with a base such as LiOHin THF/H₂O or aqueous acid to provide J5. In another embodiment, J6 canalso be coupled with J7 to provide J3.

Compounds prepared according to the schemes described above may beobtained as single enantiomers, diastereomers, or regioisomers, byenantio-, diastero-, or regiospecific synthesis, or by resolution.Compounds prepared according to the schemes above may alternately beobtained as racemic (1:1) or non-racemic (not 1:1) mixtures or asmixtures of diastereomers or regioisomers. Where racemic and non-racemicmixtures of enantiomers are obtained, single enantiomers may be isolatedusing conventional separation methods known to one skilled in the art,such as chiral chromatography, recrystallization, diastereomeric saltformation, derivatization into diastereomeric adducts,biotransformation, or enzymatic transformation. Where regioisomeric ordiastereomeric mixtures are obtained, single isomers may be separatedusing conventional methods such as chromatography or crystallization.

For starting materials requiring stereospecific amino acid chemistry,these materials were purchased as preferred stereospecific enantiomerswhich retained their specificity throughout the synthesis reactions.

The following examples are provided to further illustrate the inventionand various preferred embodiments.

EXAMPLES Chemistry

In obtaining the compounds described in the examples below and thecorresponding analytical data, the following experimental and analyticalprotocols were followed unless otherwise indicated.

Unless otherwise stated, reaction mixtures were magnetically stirred atroom temperature (rt). Where solutions were “dried,” they were generallydried over a drying agent such as Na₂SO₄ or MgSO₄. Where mixtures,solutions, and extracts were “concentrated”, they were typicallyconcentrated on a rotary evaporator under reduced pressure.

Thin-layer chromatography (TLC) was performed using Merck silica gel 60F₂₅₄ 2.5 cm×7.5 cm 250 μm or 5.0 cm×10.0 cm 250 μm pre-coated silica gelplates. Preparative thin-layer chromatography was performed using EMScience silica gel 60 F₂₅₄ 20 cm×20 cm 0.5 mm pre-coated plates with a20 cm×4 cm concentrating zone. Normal-phase flash column chromatography(FCC) was performed on silica gel (SiO₂) eluting with 2 M NH₃ inMeOH/DCM, unless otherwise noted.

Reversed-phase HPLC was performed on a Hewlett Packard HPLC Series 1100,with a Phenomenex Luna C18 (5 μm, 4.6×150 mm) column. Detection was doneat X=230, 254 and 280 nm. The gradient was 10 to 99% acetonitrile/water(0.05% trifluoroacetic acid) over 5.0 min with a flow rate of 1 mL/min.Alternatively, HPLC was performed on a Dionex APS2000 LC/MS with aPhenomenex Gemini C18 (5 μm, 30×100 mm) column, and a gradient of 5 to100% acetonitrile/water (20 mM NH₄OH) over 16.3 min, and a flow rate of30 mL/min.

Mass spectra (MS) were obtained on an Agilent series 1100 MSD equippedwith a ESI/APCI positive and negative multimode source unless otherwiseindicated. Nuclear magnetic resonance (NMR) spectra were obtained onBruker model DRX spectrometers. The format of the ¹H NMR data below is:chemical shift in ppm downfield of the tetramethylsilane reference(apparent multiplicity, coupling constant J in Hz, integration).Chemical names were generated using ChemDraw Version 6.0.2(CambridgeSoft, Cambridge, Mass.).

Example 1 [(1H-Benzoimidazole-2-carbonyl)-amino]-acetic acid

Step A: [(1H-Benzoimidazole-2-carbonyl)-amino]-acetic acid methyl ester

According to Scheme A, triethylamine (0.77 mL, 5.5 mmol) was addeddropwise to a mixture of 1H-benzoimidazole-2-carboxylic acid (0.20 g,1.2 mmol), glycine methyl ester hydrochloride (0.17 g, 1.4 mmol), HATU(0.57 g, 1.5 mmol), and DMF (10 mL). The reaction was allowed to proceedfor 16 h at 23° C. Water (25 mL) was added, and the resultingprecipitate was collected and dried (0.18 g, 61%). MS (ESI/CI): masscalcd. for C₁₁H₁₁N₃O₃, 233.2; m/z found, 234.1 [M+H]⁺. ¹H NMR (500 MHz,DMSO-d₆): 13.29 (s, 1H), 9.23 (t, J=6.1 Hz, 1H), 7.75 (d, J=7.2 Hz, 1H),7.55 (d, J=7.26 Hz, 1H), 7.31 (m, 2H), 4.08 (d, J=6.1 Hz, 2H), 3.68 (s,3H).

Step B: [(1H-Benzoimidazole-2-carbonyl)-amino]-acetic acid

A solution of LiOH.H₂O (0.090 g, 2.1 mmol) and H₂O (2 mL) was added to amixture of [(1H-benzoimidazole-2-carbonyl)-amino]-acetic acid methylester (0.10 g, 0.43 mmol) and THF (5 mL). The resulting mixture wasstirred rapidly for 30 min, then the THF was removed in vacuo. Asolution of 1M HCl (3 mL) was added, and the resulting precipitate wascollected to provide the titled compound (0.085 g, 90%). MS (ESI/CI):mass calcd. for C₁₀H₉N₃O₃, 219.2; m/z found, 220.0 [M+H]⁺. ¹H NMR (500MHz, DMSO-d₆): 12.40-12.90 (broad s, 2H), 9.06 (t, J=6.10, 6.10 Hz, 1H),7.64 (s, 1H), 7.31 (m, 2H), 3.99 (d, J=6.14 Hz, 2H).

Example 2 (S)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-propionic acid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₁H₁₁N₃O₃, 233.2; m/z found, 234.1 [M+H]⁺. ¹HNMR (600 MHz, DMSO-d₆): 13.31 (s, 1H), 12.78 (s, 1H), 9.00 (d, J=7.6 Hz,1H), 7.75 (d, J=7.7 Hz, 1H), 7.55 (d, J=7.7 Hz, 1H), 7.31 (m, 2H),4.53-4.46 (m, 1H), 1.46 (d, J=7.3 Hz, 3H).

Example 3 [(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₀H₇C₁₂N₃O₃, 287.0; m/z found, 288.0 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆): 13.66 (s, 1H), 12.76 (s, 1H), 9.23 (t, J=6.1Hz, 1H), 8.05 (s, 1H), 7.75 (s, 1H), 3.97 (d, J=6.1 Hz, 2H).

Example 4(S)-2-[(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)-amino]-propionic acid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₁H₉C₁₂N₃O₃, 302.1; m/z found, 300.0 [M−H]⁻.¹H NMR (400 MHz, DMSO-d₆): 12.30-14.40 (br. s, 2H), 9.14 (d, J=7.6 Hz,1H), 7.90 (br. s, 2H), 4.44-4.53 (m, 1H), 1.45 (d, J=7.3 Hz, 3H).

Example 5 [(6-Nitro-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₀H₈N₄O₆, 264.05; m/z found, 265.0 [M+H]⁺. ¹HNMR (400 MHz, DMSO-d₆): resonances assignable to major tautomer, 14.02(s, 0.65H), 12.78 (s, 1H), 9.34 (br t, J=6.0 Hz, 1H), 8.64 (br s, 1H),8.25 (dd, J=8.9, 1.8 Hz, 1H), 7.74 (d, J=8.8, 1 H), 3.99 (d, J=6.4 Hz,2H); resonances assignable to minor tautomer, 14.07 (s, 1H), 12.78 (s,1H), 9.34 (br t, J=6.0 Hz, 1H), 8.39 (br s, 1H), 8.18 (d, J=8.8, 1H),7.97 (d, J=9.2, 1 H), 3.99 (d, J=6.4 Hz, 2H).

Example 6[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₂H₇F₆N₃O₃, 355.2; m/z found, 354.0 [M−H]⁻.¹H NMR (400 MHz, DMSO-d₆): 1H NMR (600 MHz, DMSO-d₆): 14.39 (s, 1H),12.83 (s, 1H), 9.16 (s, 1H), 8.15 (s, 1H), 7.94-7.93 (m, 1H), 4.03 (d,J=6.1 Hz, 2H).

Example 7(S)-2-[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₃H₉F₆N₃O₃, 369.2; m/z found, 368.0 [M−H]⁻.1H NMR (500 MHz, DMSO-d₆): 9.06 (d, J=7.6 Hz, 1H), 8.18 (s, 1H), 7.91(s, 1H), 4.54 (q, J=7.23 Hz, 1H), 1.48 (d, J=7.3 Hz, 3H).

Example 8 [(5-Iodo-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₀H₈IN₃O₃, 345.1; m/z found, 343.9 [M−H]⁻. ¹HNMR (500 MHz, DMSO-d₆, mixture of tautomers): 13.50 (s, 0.51H, majortautomer), 13.41 (s, 0.48H, minor tautomer), 12.73 (s, 1H), 9.16 (t,J=6.0 Hz, 1H), 8.11 (s, 0.51H, major tautomer), 7.87 (s, 0.48H, minortautomer), 7.61 (d, J=8.7 Hz, 0.48H, minor tautomer), 7.58 (s, 1H), 7.39(d, J=8.7, 0.51H, major tautomer), 3.97 (d, J=6.1 Hz, 2H).

Example 9 [(6-Bromo-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₀H₈BrN₃O₃, 297.0; m/z found, 298.0 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, mixture of tautomers): 13.55 (m, 1H), 12.75(br s, 1H), 9.16 (br s, 1H), 7.98-7.45 (m, 3H), 4.01 (d, J=6.0 Hz, 2H).

Example 10(S)-2-[(6-Fluoro-1H-benzoimidazole-2-carbonyl)-amino]-propionic acid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₁H₁₀FN₃O₃, 251.1; m/z found, 252.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, mixture of tautomers): 9.05 (d, J=7.6 Hz, 1H),7.71-7.67 (m, 1H), 7.44 (d, J=8.4 Hz, 1H), 7.24-7.19 (m, 1H), 4.54-4.50(m, 1H), 1.48 (d, J=7.2 Hz, 3H).

Example 11(R)-2-[(6-Fluoro-1H-benzoimidazole-2-carbonyl)-amino]-propionic acid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₁H₁₀FN₃O₃, 251.1; m/z found, 252.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, mixture of tautomers): 9.04 (d, J=7.6 Hz, 1H),7.69 (br s, 1H), 7.44 (br s, 1H), 7.22 (t, J=9.2 Hz, 1H), 4.53-4.51 (m,1H), 1.48 (d, J=7.2 Hz, 3H).

Example 12 [(6-Methoxy-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₁H₁₁N₃O₄, 249.1; m/z found, 250.1 [M+H]⁺. ¹HNMR (400 MHz, DMSO-d₆, mixture of tautomers): 13.22 (br s, 1H), 12.75(br s, 1H), 9.02 (t, J=6.0 Hz, 1H), 7.65-6.94 (m, 3H), 3.99 (d, J=5.6Hz, 2H), 3.84 (s, 3H).

Example 13({5-[3-(3-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid

Step A:5-Bromo-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylicacid tert-butyl ester and6-Bromo-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylicacid tert-butyl ester

According to Scheme B, triethylamine (0.57 g, 10 mmol),4-(dimethylamino)pyridine (0.06 g, 0.5 mmol), and (Boc)₂O (2.2 g, 10.3mmol) were added to a solution of[(5-bromo-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid methyl ester(prepared in a manner analogous to EXAMPLE 1, step A) (1.6 g, 5.1 mmol)and CH₂Cl₂ (30 ml). The resulting mixture was stirred at 23° C. for 1hr. The solution was then concentrated and the residue waschromatographed (15:85 EtOAc/hexanes) to produce the titled compounds(2.3 g, 110%). MS (ESI/CI): mass calcd. for C₁₆H₁₈BrN₃O₅, 411.0; m/zfound, 412.0 [M+H]⁺.

Step B:5-[3-(3-Chloro-benzyloxy)-phenyl]-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylicacid tert-butyl ester and6-[3-(3-Chloro-benzyloxy)-phenyl]-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylicacid tert-butyl ester

[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium (0.046 g, 0.06mmol) was added to a mixture of cesium fluoride (0.19 g, 1.2 mmol),3-(3′-chlorobenzyloxyl)phenylboronic acid (0.22 g, 0.75 mmol),5-bromo-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylicacid tert-butyl ester and6-bromo-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylicacid tert-butyl ester (0.26 g, 0.63 mmol) and DME (5 mL) in a sealabletube. The reaction mixture was stirred at 80° C. for 3 h, then themixture was allowed to cool and was diluted with EtOAc (50 ml) andfiltered. The filtrate was concentrated and the residue waschromatographed (85:15 EtOAc/hexanes) to produce the titled compounds(0.22 g, 63%). MS (ESI/CI): mass calcd. for C₂₉H₂₈ClN₃O₆, 549.2; m/zfound, 550.1 [M+H]⁺.

Step C:({5-[3-(3-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid methyl ester

TFA (0.45 g, 3.9 mmol) was added to a solution of5-[3-(3-chloro-benzyloxy)-phenyl]-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylicacid tert-butyl ester,6-[3-(3-chloro-benzyloxy)-phenyl]-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylicacid tert-butyl ester (0.22 g, 0.39 mmol) and CH₂Cl₂ (2 ml). The mixturewas stirred for 1 hr and was neutralized with sat. NaHCO₃. The resultingprecipitate was collected to afford the titled compound (0.12 g, 67%).MS (ESI/CI): mass calcd. for C₂₄H₂₀ClN₃O₄, 449.1; m/z found, 450.1[M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.78-12.68(m, 1H), 9.24 (d, J=1.1 Hz, 1H), 8.06-7.53 (m, 4H), 7.52-7.37 (m, 4H),7.36-7.24 (m, 2H), 7.02 (d, J=6.7 Hz, 1H), 5.23 (s, 2H), 4.09 (d, J=6.1Hz, 2H), 3.68 (s, 3H).

Step D:({5-[3-(3-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 1,Step B. MS (ESI/CI): mass calcd. for C₂₃H₁₈ClN₃O₄, 435.1; m/z found,436.1[M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.37 (d,J=13.6 Hz, 1H), 9.06 (d, J=5.8 Hz, 1H), 8.09-7.53 (m, 4H), 7.53-7.36 (m,4H), 7.36-7.24 (m, 2H), 7.09-6.95 (m, 1H), 5.23 (s, 2H), 3.99 (d, J=6.1Hz, 2H).

Example 14({5-[3-(2-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₂₃H₁₈ClN₃O₄, 435.1; m/z found, 436.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.41 (d, J=12.9 Hz,1H), 9.12 (d, J=5.5 Hz, 1H), 8.09-7.18 (m, 9H), 7.04 (t, J=6.3, Hz, 1H),5.27 (s, 2H), 3.98 (d, J=6.1 Hz, 2H).

Example 15({5-[3-(4-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₂₃H₁₈ClN₃O₄, 435.1; m/z found, 436.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.36 (d, J=12.6 Hz,1H), 9.07 (dd, J=14.4, 6.0 Hz, 1H), 8.07-7.19 (m, 10H), 7.01 (t, J=6.2,6.2 Hz, 1H), 5.21 (s, 2H), 4.00 (d, J=6.1 Hz, 2H).

Example 16({5-[3-(3-Fluoro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₂₃H₁₈FN₃O₄, 419.1; m/z found, 420.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.37 (d, J=13.2 Hz,1H), 9.08 (s, 1H), 8.12-7.22 (m, 9H), 7.21-7.12 (m, 1H), 7.02 (d, J=7.4Hz, 1H), 5.24 (s, 2H), 4.00 (d, J=6.1 Hz, 2H).

Example 17({5-[3-(2-Fluoro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₂₃H₁₈FN₃O₄, 419.1; m/z found, 420.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.36 (d, J=13.7 Hz,1H), 9.35-8.77 (m, 1H), 8.09-7.16 (m, 10H), 7.03 (t, J=6.1, 6.1 Hz, 1H),5.25 (s, 2H), 3.99 (d, J=6.1 Hz, 2H).

Example 18{[5-(4-Phenoxy-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-acetic acid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₂₂H₁₇N₃O₄, 387.1; m/z found, 388.1 [M+H]⁺. ¹HNMR (400 MHz, DMSO-d₆, tautomeric broadening): 9.14 (s, 1H), 7.82 (s,1H), 7.77-7.68 (m, 3H), 7.60 (dd, J=8.5, 1.7 Hz, 1H), 7.43 (dd, J=8.5,7.4 Hz, 2H), 7.23-7.14 (m, 1H), 7.14-7.04 (m, 4H), 3.99 (d, J=6.1 Hz,2H).

Example 19{[5-(3-Benzyloxy-5-fluoro-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₂₃H₁₈FN₃O₄, 419.1; m/z found, 420.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.45 (d, J=25.0 Hz,1H), 9.28-8.91 (m, 1H), 8.09-7.56 (m, 3H), 7.50 (d, J=7.1 Hz, 2H), 7.42(t, J=7.3, 7.3 Hz, 2H), 7.39-7.31 (m, 1H), 7.25-7.07 (m, 2H), 6.97-6.85(m, 1H), 5.23 (s, 2H), 3.99 (d, J=6.1 Hz, 2H).

Example 20 [(5-Quinolin-3-yl-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₁₉H₁₄N₄O₃, 346.1; m/z found, 347.1 [M+H]⁺. ¹HNMR (400 MHz, DMSO-d₆, tautomeric broadening): 9.37 (d, J=2.2 Hz, 1H),9.13 (t, J=6.1, 6.1 Hz, 1H), 8.81 (d, J=1.8 Hz, 1H), 8.21-8.06 (m, 3H),7.90-7.78 (m, 3H), 7.77-7.63 (m, 1H), 4.02 (d, J=6.1 Hz, 2H).

Example 21({5-[3-Chloro-4-(3-chloro-benzyloxy)phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₂₃H₁₇Cl₂N₃O₄, 469.1; m/z found, 470.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.41 (d, J=17.1 Hz,1H), 9.12 (d, J=6.1 Hz, 1H), 8.06-7.52 (m, 6H), 7.53-7.38 (m, 3H),7.37-7.27 (m, 1H), 5.30 (s, 2H), 3.99 (d, J=6.1 Hz, 2H).

Example 22({5-[4-(3-Chloro-benzyloxy)-3,5-dimethyl-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 13. ¹HNMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.40 (br. s, 1H), 13.36(br. s, 1H), 9.06-9.15 (m, 1H), 7.34-7.95 (m, 9H), 4.87 (s, 2H), 3.99(d, J=6.07 Hz, 2H), 2.33 (s, 6H).

Example 23{[5-(3-Methoxy-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-acetic acid

The titled compound was prepared in a manner analogous to EXAMPLE 13. ¹HNMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.36 (br. s, 1H), 9.07(br. m, 1H), 6.95-8.10 (m, 7H), 4.00 (d, J=6.09 Hz, 1H), 3.84 (s, 3H).

Example 24{[5-(5-Benzylcarbamoyl-2-fluoro-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₁₇H₁₅N₃O₄, 446.4; m/z found, 447.1 [M+H]⁺. ¹HNMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.45 (d, J=7.60 Hz, 1H),12.2-13.0 (br. s, 1H), 9.05-9.2 (m, 2H), 7.15-8.20 (m, 11H), 4.51 (d,J=5.9 Hz, 2H), 4.00 (d, J=6.1 Hz, 2H).

Example 25[(5-Naphthalen-2-yl-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₂₀H₁₅N₃O₃, 345.4; m/z found, 346.1 [M+H]⁺. ¹HNMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.41-13.52 (m, 1H),9.09-9.20 (m, 1H), 8.26 (br. s, 1H), 7.60-8.10 (m, 7H), 7.45-7.60 (m,2H), 4.00 (d, J=6.1 Hz, 2H).

Example 26{[5-(4-Propoxy-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-acetic acid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₁₉H₁₉N₃O₄, 353.4; m/z found, 354.1 [M+H]⁺. ¹HNMR (400 MHz, DMSO-d₆, tautomeric broadening): 12.90-13.80 (br s, 1H),9.08-9.16 (m, 1H), 7.50-8.10 (m, 3H), 7.10-7.45 (m, 3H), 6.91-6.97 (m,1H), 6.45-6.64 (br. m, 1H), 3.96-4.06 (m, 4H), 2.54-2.57 (m, 2H),1.72-1.83 (m, 3H).

Example 27{[5-(4-Chloro-3-methyl-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₁₇H₁₄ClN₃O₄, 343.8; m/z found, 344.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.35-13.53 (br. m,1H), 9.00-9.22 (m, 1H), 7.46-8.42 (m, 6H), 4.00 (d, J=6.1 Hz, 2H), 2.42(s, 3H).

Example 28{[5-(5-Chloro-2-fluoro-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 13. MS(ESI/CI): mass calcd. for C₁₆H₁₁ClFN₃O₄, 347.7; m/z found, 348.0 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.45-13.53 (m, 1H),9.13-9.22 (m, 1H), 7.35-7.96 (m, 6H), 3.98 (d, J=6.1 Hz).

Example 29 [(5-Benzoylamino-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid

Step A: 5-Benzoylamino-1H-benzoimidazole-2-carboxylic acid methyl ester

According to Scheme C, DIPEA (0.45 mL, 2.6 mmol) was added to a solutionof 5-amino-1H-benzoimidazole-2-carboxylic acid methyl ester (0.200 g,1.04 mmol) in THF (5 mL) at 0° C., followed by benzoyl chloride (0.154g, 1.09 mmol). After 2 h, the reaction was quenched with water (6 mL),the THF was evaporated, and the resulting aqueous layer extracted withEtOAc (3×15 mL). The combined organic layers were washed with brine (15mL), dried, and concentrated to yield the desired product (0.272 g,89%). MS (ESI/CI): mass calcd. for C₁₆H₁₃N₃O₃, 295.10; m/z found, 296.1[M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): 13.50 (s, 0.3H), 13.44 (s, 0.7H),10.41 (s, 0.7H), 10.33 (s, 0.3H), 8.30 (d, J=2.0 Hz, 0.7H), 8.26 (d,J=1.2 Hz, 0.3H), 8.01-7.96 (m, 2H), 7.76-7.70 (m, 1H), 7.63-7.51 (m,4H), 3.96-3.94 (m, 3H).

Step B: 5-Benzoylamino-1H-benzoimidazole-2-carboxylic acid

LiOH.H₂O (0.23 g, 5.4 mmol) was added to a solution of5-benzoylamino-1H-benzoimidazole-2-carboxylic acid methyl ester (0.25 g,0.85 mmol) and THF (6 mL) at rt, followed by water (2 mL). Afterstirring for 1 h the THF was evaporated and HCl (1 M, 10 mL) was added.The resulting precipitate was dried to yield the desired compound (0.210g, 88%). MS (ESI): mass calcd. for C₁₆H₁₁N₃O₃, 281.08; m/z found, 282.1[M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): 10.42 (s, 1H), 8.30 (d, J=1.2 Hz,1H), 7.99 (dt, J=6.8, 1.6 Hz, 2H), 7.71-7.52 (m, 5H).

Step C: [(5-Benzoylamino-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₇H₁₄N₄O₄, 338.10; m/z found, 339.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆): 10.36 (s, 1H), 9.08 (t, J=6.0 Hz, 1H), 8.27(t, J=1.2 Hz, 1H), 8.00 (t, J=1.3 Hz, 1H), 7.98 (t, J=1.8 Hz, 1H),7.69-7.49 (m, 5H), 3.98 (d, J=6.1 Hz, 2H).

Example 30[(6-Benzenesulfonylamino-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid

Step A: 5-Benzenesulfonylamino-1H-benzoimidazole-2-carboxylic acidmethyl ester

According to Scheme C, DIPEA (0.45 mL, 2.6 mmol) was added to a solutionof 5-amino-1H-benzoimidazole-2-carboxylic acid methyl ester (0.200 g,1.04 mmol) and THF (5 mL) at 0° C., followed by benzenesulfonyl chloride(0.193 g, 1.09 mmol). After 3.5 h the reaction was quenched with water(5 mL), the THF was evaporated, and the resulting aqueous layer wasextracted with EtOAc (3×10 mL). The combined organic layers were washedwith brine (10 mL), toluene (2 mL) was added, and the solution wasconcentrated to yield the titled compound (0.335 g, 97%). MS (ESI/CI):mass calcd. for C₁₅H₁₃N₃O₄S, 331.06; m/z found, 332.1 [M+H]⁺. ¹H NMR(400 MHz, DMSO-d₆): 13.36 (br s, 1H), 10.47 (br s, 1H), 7.73 (d, J=7.6Hz, 2H), 7.62-7.50 (m, 4H), 7.34 (br s, 1H), 7.07 (br s, 1H), 3.91 (s,3H).

Step B:[(6-Benzenesulfonylamino-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid

The titled compound was prepared in a manner analogous to EXAMPLE 29,Steps B-C. MS (ESI/CI): mass calcd. for C₁₆H₁₄N₄O₅S, 374.07; m/z found,375.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): 10.24 (s, 1H), 9.03 (t, J=6.2Hz, 1H), 7.73-7.71 (m, 1H), 7.70 (t, J=1.8, 1H), 7.61-7.44 (m, 4H), 7.33(d, J=1.8 Hz, 1H), 7.04 (dd, J=8.8, 2.0 Hz, 1H), 3.94 (d, J=6.1 Hz, 2H).

Example 31 [(6-Benzylamino-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid

Step A: 6-Benzylamino-1H-benzoimidazole-2-carboxylic acid methyl ester

According to Scheme D, Benzaldehyde (0.11 mL, 1.0 mmol) and NaBH(OAc)₃(0.309 g, 1.46 mmol) were added to a solution of5-amino-1H-benzoimidazole-2-carboxylic acid methyl ester (0.200 g, 1.04mmol) and 1,2-dichloroethane (4 mL). After 8 h, additional benzaldehyde(0.010 mL, 0.10 mmol) was added; after an additional 15 h, NaBH(OAc)₃(0.221 g, 1.04 mmol) was added to the reaction. After 5 h the reactionwas quenched with sat. aq. NaHCO₃ (10 mL) and extracted with EtOAc (3×15mL). The combined organic layers were washed with brine (15 mL), dried,and concentrated. The resulting residue was chromatographed (35-75%EtOAc/hexanes) to yield the titled compound (0.268 g, 91%). MS (ESI/CI):mass calcd. for C₁₆H₁₅N₃O₂, 281.12; m/z found, 282.1 [M+H]⁺. ¹H NMR (400MHz, DMSO-d₆): 12.78 (s, 1H), 7.44-7.36 (m, 3H), 7.33 (t, J=7.6 Hz, 2H),7.23 (t, J=7.2 Hz, 1H), 6.78 (d, J=8.5 Hz, 1H), 6.56 (t, J=5.5 Hz, 1H),6.39 (s, 1H), 4.30 (d, J=5.8 Hz, 2H), 3.87 (s, 3H).

Step B: [(6-Benzylamino-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid

The titled compound was prepared in a manner analogous to EXAMPLE 29,Step B-C. MS (ESI/CI): mass calcd. for C₁₇H₁₆N₄O₃, 324.12; m/z found,325.1 [M+H]⁺. ¹H NMR (400 MHz, CD₃OD): 7.62 (d, J=6.8 Hz, 1H), 7.47-7.28(m, 5H), 7.20-7.08 (m, 2H), 4.51 (s, 2H), 4.17 (s, 2H).

Example 32 1-(1H-Benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid

Step A: Preparation of2-chloro-1-(2-trimethylsilanyl-ethoxymethyl)-1H-benzoimidazole

According to Scheme E, a mixture of NaH (60% dispersion in oil, 0.40 g,9.8 mmol) and THF (10 mL) was cooled to 0° C., then solid2-chlorobenzoimidazole (1.0 g, 6.5 mmol) was added portion wise over 10min. The resulting mixture was stirred at 0° C. for 1 h, then2-trimethylsilylethoxymethyl chloride (1.5 mL, 8.5 mmol) was added. Thereaction mixture was allowed to warm to 23° C. and was stirred 16 h. Themixture was carefully poured over ice (ca. 200 g) and then was extractedwith Et₂O (3×100 mL). The combined organic extracts were dried andconcentrated, and the residue was chromatographed (1:99 to 15:85EtOAc/hexanes) to provide the titled compound, which has been previouslydescribed: PCT Int. Appl. (2005), 465 pp. CODEN: PIXXD2 WO 2005012297 A120050210 CAN 142:219286 AN 2005:120926.

Step B:1-[1-(2-Trimethylsilanyl-ethoxymethyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid ethyl ester

A mixture of2-chloro-1-(2-trimethylsilanyl-ethoxymethyl)-1H-benzoimidazole (0.34 g,1.2 mmol), ethyl pryazole-4-carboxylate (0.24 g, 1.7 mmol), cesiumcarbonate (0.78 g, 2.4 mmol), and anhydrous DMF (2.5 mL) was stirred at100° C. for 5 h. The mixture was allowed to cool to 23° C. and wasdiluted with EtOAc, then filtered through a pad of silica gel. Theresulting solution was concentrated and the residue was chromatographed(5:95 to 40:60 EtOAc/hexanes) to providing the titled compound (0.36 g,77%). 1H NMR (500 MHz, CDCl₃): 8.88 (s, 1H), 8.18 (s, 1H), 7.77-7.69 (m,1H), 7.60-7.50 (m, 1H), 7.40-7.30 (m, 2H), 6.03 (s, 2H), 4.34 (q, J=7.1Hz, 2H), 3.57-3.50 (m, 2H), 1.37 (t, J=7.1, Hz, 3H), 0.87-0.80 (m, 2H),−0.11 (s, 9H).

Step C: 1-(1H-Benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid ethylester hydrochloride

A solution of HCl and dioxane (4M, 2 mL, 8 mmol) was added to a mixtureof1-[1-(2-trimethylsilanyl-ethoxymethyl)-1H-benzoimidazol-2-yl]-1H-pyrrole-3-carboxylicacid ethyl ester (0.30 g, 0.78 mmol) and EtOH (4 mL). The reactionmixture was heated to reflux for 30 min, then cooled to 23° C. Et₂O wasadded (20 mL), and the mixture was cooled to 0° C. for 10 min. Theresulting precipitate was collected by filtration and washed well withEt₂O to afford the titled compound (0.18 g, 91%). MS (ESI/CI): masscalcd. for C₁₃H₁₂N₄O₂, 256.3; m/z found, 257.1 [M+H]⁺. ¹H NMR (400 MHz,DMSO-d₆): 8.96 (s, 1H), 8.33 (s, 1H), 7.56 (s, 2H), 7.28-7.21 (m, 2H),4.30 (q, J=7.1 Hz, 2H), 1.32 (t, J=7.1 Hz, 3H)

Step D: 1-(1H-Benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid

A solution of LiOH and H₂O (1.0 M, 1.0 mL, 1.0 mmol) was added to amixture of 1-(1H-benzoimidazol-2-yl)-1H-pyrrole-3-carboxylic acid ethylester hydrochloride (0.040 g, 0.16 mmol) and THF (2.0 mL), and thereaction mixture was stirred at 23° C. for 16 h. The THF was removed invacuo and then aqueous HCl (1.0 M, 2 mL, 2 mmol) was added at 0° C. Theresulting precipitate was collected and washed with water to give thetitled compound (0.033 g, 90%). MS (ESI/CI): mass calcd. for C₁₁H₈N₄O₂,228.2; m/z found, 229.0 [M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆): 13.32 (s,1H), 13.00-12.86 (br. s, 1H), 8.90 (d, J=0.6 Hz, 1H), 8.28 (d, J=0.6 Hz,1H), 7.64 (d, J=4.6 Hz, 1H), 7.49 (d, J=5.5 Hz, 1H), 7.28-7.20 (m, 2H).

Example 331-(5,6-Dichloro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₁H₆Cl₂N₄O₂, 297.1; m/z found, 296.0 [M−H]⁻.¹H NMR (500 MHz, DMSO-d₆): 14.18-12.52 (br. s, 2H), 8.89 (d, J=0.5 Hz,1H), 8.31 (d, J=0.5 Hz, 1H), 7.80 (s, 2H).

Example 341-(5-Trifluoromethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₂H₇F₃N₄O₂, 296.2; m/z found, 295.0 [M−H]⁻.¹H NMR (500 MHz, DMSO-d₆): 14.44-12.32 (br. s, 2H), 8.94 (d, J=0.5 Hz,1H), 8.33 (d, J=0.5 Hz, 1H), 7.96-7.83 (br. s, 1H), 7.75 (br. d, 1H),7.58 (dd, J=8.49, 1.41 Hz, 1H).

Example 351-(5-Chloro-6-fluoro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₁H₆ClFN₄O₂, 280.7; m/z found, 279.0 [M−H]⁻.¹H NMR (500 MHz, DMSO-d₆): 14.21-12.25 (br. S, 2H), 8.88 (d, J=0.6 Hz,1H), 8.30 (d, J=0.6 Hz, 1H), 7.81-7.67 (br. s, 1H), 7.65-7.52 (br. s,1H).

Example 361-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₃H₁₂N₄O₂, 256.3; m/z found, 257.1 [M+H]⁺. ¹HNMR (500 MHz, DMSO-d₆): 13.16-12.81 (m, 2H), 8.85 (d, J=0.6 Hz, 1H),8.25 (d, J=0.6 Hz, 1H), 7.43-7.21 (br. s, 2H), 2.31 (s, 6H)

Example 37 1-(5-Bromo-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₁H₇BrN₄O₂, 306.0; m/z found, 307.0 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆): 8.82 (d, J=0.5 Hz, 1H), 8.22 (s, 1H), 7.67(d, J=1.2 Hz, 1H), 7.45 (d, J=8.5 Hz, 1H), 7.32 (dd, J=8.5, 1.9 Hz, 1H).

Example 38 1-(5-Methoxy-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₂H₁₀N₄O₃, 258.2; m/z found, 259.1[M+H]⁺. ¹HNMR (500 MHz, DMSO-d₆, tautomeric mixture): 13.16 (s, 1H), 12.91 (s,1H), 8.84 (s, 1H), 8.26 (s, 1H), 6.83-7.54 (m, 3H), 3.80 (s, 3H).

Example 391-(4-Chloro-6-trifluoromethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₂H₆ClF₃N₄O₂, 330.7; m/z found, 329.0 [M−H]⁻.¹H NMR (500 MHz, DMSO-d₆): 13.90-14.50 (br. s, 1H), 12.75-13.45 (br. s,1H), 8.95 (s, 1H), 8.36 (s, 1H), 7.72 (s, 1H), 7.70 (s, 1H).

Example 401-(5,6-Dimethoxy-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₃H₁₂N₄O₄, 288.3; m/z found, 289.1 [M+H]⁺. ¹HNMR (500 MHz, DMSO-d₆): 8.81 (s, 1H), 8.25 (s, 1H), 7.09 (s, 2H), 3.80(s, 6H).

Example 411-(4,5-Dimethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₃H₁₂N₄O₂, 256.3; m/z found, 257.2 [M+H]⁺. ¹HNMR (500 MHz, DMSO-d₆, tautomeric mixture): 12.60-13.30 (br. m, 2H),8.83-8.90 (m, 1H), 8.23-8.29 (m, 1H), 7.0-7.35 (m, 2H), 2.47 (s, 3H),2.33 (s, 3H).

Example 421-(5-Trifluoromethoxy-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid

Prepared in the same manner analogous to EXAMPLE 32. MS (ESI/CI): Masscalcd. for C₁₂H₇F₃N₄O₃ 312.0. m/z found: 313.1 [M+H]⁺. ¹H NMR (400 MHz,DMSO-d₆): 8.91 (s, 1H), 8.31 (s, 1H), 7.83-7.41 (m, 2H), 7.30-7.21 (m,1H).

Example 431-{5-[3-(3-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid

Step A:1-{5-[3-(3-Choro-benzyloxy)-phenyl]-1-(2-trimethylsilanyl-ethoxymethyl)-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid ethyl ester and1-{6-[3-(3-Choro-benzyloxy)-phenyl]-1-(2-trimethylsilanyl-ethoxymethyl)-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid ethyl ester

According to Scheme F, [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium (0.12 g, 0.16 mmol) was added to a mixture of cesium fluoride(0.33 g 2.2 mmol), 3-(3′-chlorobenzyloxyl)phenylboronic acid (0.37 g,1.3 mmol),1-[5-Bromo-1-(2-trimethylsilanyl-ethoxymethyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid ethyl ester and1-[6-Bromo-1-(2-trimethylsilanyl-ethoxymethyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid ethyl ester (mixture of regioisomers prepared in a manner analogousto EXAMPLE 31, Step A. MS (ESI/CI): Mass calcd. for C₁₉H₂₅BrN₄O₃Si,464.1; m/z found, 465.1), (0.5 g, 1.1 mmol), and DME (5 ml) in asealable tube. The reaction was stirred at 80° C. After 3 h, the mixturewas cooled to rt, then was diluted with EtOAc (50 ml) and filtered. Thefiltrate was concentrated and the residue was chromatographed (15:85EtOAc/hexanes) to yield the titled compounds as a regioisomeric mixture(0.47 g, 72%). MS (ESI/CI): mass calcd. for C₃₂H₃₅ClN₄O₄Si, 602.2; m/zfound, 603.2 [M+H]⁺.

Step B:1-{5-[3-(3-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 32,Steps C-D. MS (ESI/CI): mass calcd. for C₂₄H₁₇ClN₄O₃ 444.1; m/z found,445.1 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) 8.93 (d, J=0.5 Hz, 1H), 8.32 (s,1H), 7.84-7.79 (m, 1H), 7.68-7.63 (m, 1H), 7.60-7.55 (m, 2H), 7.52-7.37(m, 4H), 7.36-7.27 (m, 2H), 7.03 (dd, J=7.8, 2.1 Hz, 1H), 5.26 (s, 2H).

Example 441-{5-[3-(2-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 43. MS(ESI/CI): mass calcd. for C₂₄H₁₇ClN₄O₃, 444.1; m/z found, 445.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆) 8.91 (s, 1H), 8.30 (s, 1H), 7.80 (s, 1H),7.72-7.60 (m, 2H), 7.59-7.48 (m, 2H), 7.48-7.36 (m, 3H), 7.36-7.25 (m,2H), 7.02 (dd, J=8.1, 1.9 Hz, 1H), 5.27 (s, 2H).

Example 451-{5-[3-(4-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 43. MS(ESI/CI): mass calcd. for C₂₄H₁₇ClN₄O₃, 444.1; m/z found, 445.1 [M+H]⁺.¹H NMR (600 MHz, DMSO-d₆). 8.91 (s, 1H), 8.30 (s, 1H), 7.98-7.50 (m,5H), 7.50-7.44 (m, 2H), 7.43-7.35 (m, 1H), 7.35-7.22 (m, 2H), 7.00 (s,1H), 5.22 (s, 2H).

Example 461-[5-(3-Benzyloxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 43. MS(ESI/CI): mass calcd. for C₂₄H₁₈N₄O₃, 410.1; m/z found, 411.2 [M+H]⁺. ¹HNMR (400 MHz, DMSO-d₆) 8.99 (s, 1H), 8.38 (s, 1H), 8.02-7.28 (m, 11H),7.09 (dd, J=8.1, 1.9 Hz, 1H), 5.31 (s, 2H).

Example 471-[5-(4-Benzyloxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 43. MS(ESI/CI): mass calcd. for C₂₄H₁₈N₄O₃, 410.1; m/z found; 411.2 [M+H]⁺. ¹HNMR (400 MHz, DMSO-d₆) 9.00 (s, 1H), 8.39 (s, 1H), 7.81-7.39 (m, 10H),7.20 (d, J=8.8 Hz, 2H), 5.26 (s, 2H).

Example 481-[5-(3-Trifluoromethyl-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 43. MS(ESI/CI): mass calcd. for C₁₈H₁₁F₃N₄O₂, 372.1; m/z found, 373.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆). 8.91 (s, 1H), 8.31 (s, 1H), 8.11-7.79 (m,3H), 7.78-7.52 (m, 4H).

Example 491-[5-(3,4-Dichloro-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 43. MS(ESI/CI): Mass calcd. for C₁₇H₁₀Cl₂N₄O₂ 372.0. m/z found: 373.0 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆): 9.01 (s, 1H), 8.41 (s, 1H), 8.06 (s, 1H),7.95 (s, 1H), 7.81 (d, J=1.2 Hz, 2H), 7.74 (d, J=8.4 Hz, 1H), 7.69 (dd,J=8.5, 1.7 Hz, 1H).

Example 50 (R)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-propionic acid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₁H₁₁N₃O₃, 233.2; m/z found, 234.1 [M+H]⁺. ¹HNMR (600 MHz, DMSO-d₆): 13.31 (s, 1H), 12.78 (s, 1H), 9.00 (d, J=7.6 Hz,1H), 7.75 (d, J=7.7 Hz, 1H), 7.55 (d, J=7.7 Hz, 1H), 7.31 (m, 2H),4.46-4.53 (m, 1H), 1.46 (d, J=7.3 Hz, 3H).

Example 51(R)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-3-hydroxy-propionic acid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₁H₁₁N₃O₃, 249.2; m/z found, 250.1 [M+H]⁺. ¹HNMR (500 MHz, DMSO-d₆): 8.74 (d, J=8.0 Hz, 1H), 7.66-7.72 (m, 2H),7.34-7.40 (m, 2H), 4.51-4.56 (m, 1H), 3.89-3.95 (m, 2H), 3.81-3.86 (m,2H).

Example 52(R)-2-[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₃H₉F₆N₃O₃, 369.2; m/z found, 368.0 [M−H]⁻.¹H NMR (500 MHz, DMSO-d₆): 12.20-15.00 (br. s, 1H), 9.08 (d, J=7.6 Hz,1H), 8.18 (s, 1H), 7.92 (s, 1H), 4.55 (q, J=7.3 Hz, 1H), 1.48 (d, J=7.3Hz, 3H).

Example 53(R)-2-[(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)-amino]-propionic acid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₁H₉Cl₂N₃O₃, 302.1; m/z found, 301.9 [M−H]⁻.¹H NMR (500 MHz, DMSO-d₆): 13.45-13.90 (br. s, 1H), 12.60-13.00 (m, 1H),9.13 (d, J=7.6 Hz, 1H), 8.03 (br. s, 1H), 7.76 (br. s, 1H), 4.44-4.55(m, 1H), 1.45 (d, J=7.3 Hz, 3H).

Example 54 (R)-2-[(5-Iodo-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid

The titled compound was prepared in a manner analogous to EXAMPLE 1. MS(ESI/CI): mass calcd. for C₁₁H₁₀IN₃O₃, 359.1; m/z found, 357.9 [M−H]⁻.¹H NMR (500 MHz, DMSO-d₆, tautomeric mixture): 13.34-13.51 (br. m, 1H),12.76 (s, 1H), 9.04 (d, J=7.2 Hz, 1H), 7.85-8.16 (br. m, 1H), 7.35-7.65(br. m, 2H), 4.44-4.52 (m, 1H), 1.44 (d, J=7.3 Hz, 3H).

Example 551-(5-Bromo-1H-benzoimidazol-2-yl)-3-trifluoromethyl-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₂H₆BrF₃N₄O₂, 374.0; m/z found, 375.0 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆): 13.69 (br s, 1H), 9.09 (s, 1H), 7.79 (br s,1H), 7.55 (br s, 1H), 7.43 (dd, J=8.4, 1.6 Hz, 1H).

Example 561-(5,6-dichloro-1H-benzoimidazol-2-yl)-3-trifluoromethyl-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₂H₅Cl₂F₃N₄O₂, 365.1; m/z found, 363.0[M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆): 13.25-14.30 (br. s, 2H), 9.10 (s,1H), 7.87 (br. s, 2H).

Example 571-(5-Bromo-1H-benzoimidazol-2-yl)-3,5-dimethyl-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 32. MS(ESI/CI): mass calcd. for C₁₃H₁₁BrN₄O₂, 334.0; m/z found, 335.0 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆): 13.21 (br s, 1H), 12.77 (br s, 1H), 7.73 (brs, 1H), 7.51 (br s, 1H), 7.36 (dd, J=8.4, 1.6 Hz, 1H), 2.98 (s, 3H),2.46 (s, 3H).

Example 581-(5,6-Dichloro-1H-benzoimidazol-2-yl)-3,5-dimethyl-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 32 andpurified by preparatory HPLC. MS (ESI/CI): mass calcd. forC₁₃H₁₁Cl₂N₄O₂, 325.2; m/z found, 327.1 [M+H]⁺. ¹H NMR (500 MHz,DMSO-d₆): 7.79 (s, 2H), 2.98 (s, 3H), 2.46 (s, 3H).

Example 591-[5-(4-Hydroxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 43. MS(ESI/CI): mass calcd. for C₁₇H₁₂N₄O₃, 320.3; m/z found, 321.1 [M+H]⁺. ¹HNMR (600 MHz, DMSO-d₆): 12.52-13.80 (br. s, 1H), 9.25-10.05 (br s, 1H),8.84 (s, 1H), 8.25 (s, 1H), 7.43-7.80 (m, 5H), 6.86 (d, J=8.6, 2H).

Example 601-[5-(3-Hydroxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to EXAMPLE 43. MS(ESI/CI): mass calcd. for C₁₇H₁₂N₄O₃, 320.3; m/z found, 321.1 [M+H]⁺. ¹HNMR (600 MHz, DMSO-d₆): 12.50-13.56 (br. m, 2H), 9.54 (br. s, 1H), 8.91(s, 1H), 8.30 (s, 1H), 7.45-7.88 (br. m, 3H), 7.26 (t, J=7.8 Hz, 1H),7.04-7.14 (m, 2H), 6.75 (dd, J=8.0, 1.7 Hz, 1H).

Example 61 1-(5-Chloro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to Example 32. MS(ESI/CI): mass calcd. for C₁₁H₇ClN₄O₂, 262.0; m/z found, 263.0 [M+H]⁺.¹H NMR (400 MHz, CD₃OD, tautomeric broadening): 8.89 (s, 1H), 8.17 (s,1H), 7.67-7.44 (m, 2H), 7.26 (dd, J=8.6. 1.9 Hz, 1H).

Example 621-(5-Bromo-6,7-dimethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid

The titled compound was prepared in a manner analogous to Example 32. MS(ESI/CI): mass calcd. for C₁₁H₇ClN₄O₂, 334.0; m/z found, 335.0 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆, tautomeric broadening): 13.51-12.68 (m, 2H),8.88 (s, 1H), 8.29 (s, 1H), 7.80-7.40 (m, 1H), 2.56 (s, 3H), 2.40 (s,3H).

Biological Protocols: Expression and Purification of PHD2₁₈₁₋₄₁₇

The human PHD2 expression construct containing amino acids 181-417 ofGenBank Accession ID NM_(—)022051 was cloned into a pBAD vector(Invitrogen), incorporating both an N-terminal histidine tag and aSmt3-tag, both of which are cleaved by Ulp1. Protein production wasachieved by expression in BL21 cells grown in Terrific Broth containing100 μg/ml ampicillin. Cell cultures were inoculated at 37° C. and grownto an OD₆₀₀ of 0.8. Cultures were induced with 0.1% arabinose and grownovernight at 20° C. with continuous shaking at 225 rpm. Cells were thenharvested by centrifugation and stored at −80° C. Cell pellets weresuspended in Buffer A (50 mM Tris-HCl pH 7.2, 100 mM NaCl, 100 mML-arginine, 1 mM TCEP, 0.05% (w/v) NP-40, 50 mM imidazole) followed bythe addition of lysozyme and benzonase. Cells were lysed by sonicationand the lysate was cleared by centrifugation (15,000 rpm, 90 min, 4°C.). The protein was purified by nickel affinity chromatography using aHisTrap Crude FF column (GE Healthcare). Samples were eluted in Buffer Awith a 50-200 mM imidazole gradient. Cleavage of the Smt tag with Ulp1protease was achieved via overnight incubation with dialyzing againstBuffer A. The PHD2₁₈₁₋₄₁₇ sample was then passed over a second HisTrapCrude FF column (GE Healthcare) to remove uncleaved protein. Theflow-through was then dialyzed into 50 mM MES pH 6.0, 1 mM TCEP, 5 mMNaCl for ion exchange chromatography on a HiTrap SP Cation Exchangecolumn (GE Healthcare). The PHD2₁₈₁₋₄₁₇ protein was eluted with a 0-0.2M NaCl gradient. Fractions were pooled for further purification by sizeexclusion chromatography over a Superdex 75 Size Exclusion Column (GEHealthcare). Final protein was concentrated to 4 mg/ml and dialyzed in10 mM PIPES pH 7.0, 100 mM NaCl, 0.5 mM TCEP. The protein was determinedto have a purity of >95% by gel electrophoresis.

Enzyme Activity Assay

The PHD enzymatic assay was performed in 0.5 ml of reaction mixturecontaining the following: purified PHD2₁₈₁₋₄₁₇ polypeptide (3 μg),synthetic HIF-1α peptide comprising residues[KNPFSTGDTDLDLEMLAPYIPMDDDFQLRSFDQLS] (10 μM, California PeptideResearch Inc., Napa, Calif.), and [5-¹⁴C]-2-oxoglutaric acid (50mCi/mmol, Moravek Chemicals, Brea, Calif.) in reaction buffer (40 mMTris-HCl, pH 7.5, 0.4 mg/ml catalase, 0.5 mM DTT, 1 mM ascorbate) for 10minutes. The reaction was stopped by addition of 50 μl of 70 mM H₃PO₄and 50 μl of 500 mM NaH₂PO₄, pH 3.2. Detection of [¹⁴C]-succinic acidwas achieved by separating from [5-¹⁴C]-2-oxoglutaric acid by incubatingthe reaction mixture with 100 μl of 0.16 M DNP prepared in 30%perchloric acid. Next, 50 μl of unlabeled 20 mM 2-oxoglutaric acid/20 mMsuccinic acid, serving as carrier for the radioactivity, was added tothe mixture, and was allowed to proceed for 30 minutes at roomtemperature. The reaction was then incubated with 50 μl of 1 M2-oxoglutaric acid for 30 additional minutes at room temperature toprecipitate the excess DNP. The reaction was then centrifuged at 2800×gfor 10 minutes at room temperature to separate [¹⁴C]-succinic acid inthe supernatant from the precipitated [¹⁴C]-dinitrophenylhydrazone.Fractions of the supernatant (400 μl) were counted using a beta counter(Beckman Coulter, Fullerton, Calif.). Inhibition of PHD2₁₈₁₋₄₁₇ activitywas measured as a decrease in [¹⁴C]-succinic acid production. The IC₅₀values were estimated by fitting the data to a three-parameter logisticfunction using GraphPad Prism, version 4.02 (Graph Pad Software, SanDiego, Calif.).

Cellular Assay

Hep-3B cells (ATCC, Manassas, Va.) were plated in 96-well plates at20,000 cells per well in 100 μl of DMEM containing 10% fetal bovineserum, 1% non-essential amino acids, 50 IU/mL of penicillin and 50 μg/mLof streptomycin (all cell culture reagents from Invitrogen, Carlsbad,Calif.). Twenty-four hours after plating, compounds were added andincubated for an additional 24 hours. All compounds were tested undersaturating conditions with final compound concentrations at 100 μM, withthe exception of ex. #50, 51, and 53. These three compounds were testedat 10 μM. Fifty microliters of the supernatant was then transferred to ahuman Hypoxia assay kit (Meso-Scale Discovery, Gaithersburg, Md.).Erythropoietin in the supernatant was detected according to themanufacturer's instructions as follows. EPO detection plates wereblocked with 3% BSA in PBS overnight and 50 μl of the supernatant wasincubated at room temperature in an orbital shaker for 2 h. Twenty-fivemicroliters of 0.5 μg/ml anti-EPO detection antibody was added for 2hours at room temperature in an orbital shaker. After 3 washes in PBS,150 μl of 1× read buffer is added and the plate is then read on the MSDSECTOR instrument. Data was analyzed by determining the percent of EPOsecretion in the presence of 10 μM or 100 μM compound relative to anassay control compound,7-[(4-Chloro-phenyl)-(5-methyl-isoxazol-3-ylamino)-methyl]-quinolin-8-ol.

While the invention has been illustrated by reference to exemplary andpreferred embodiments, it will be understood that the invention isintended not to be limited to the foregoing detailed description.

What is claimed is:
 1. A compound of the formula:

or a pharmaceutically acceptable salt, pharmaceutically acceptableprodrug, or pharmaceutically active metabolite thereof, wherein X is anoptionally substituted heteroaryl selected from pyrazole, pyridine,pyrimidine, and pyridazine; A is a bond or optionally substitutedmethylene; each Z is independently C or N; n is 0-4; and R¹ isindependently selected from hydrogen, halogen, nitro, —C₁₋₄alkyl,alkoxy, cycloalkoxy, CON(R^(y))(R^(z)), trifluoromethyl,trifluoromethoxy, N(R^(y))R^(z) (wherein R^(y) and R^(z) areindependently hydrogen, aryl, arylsulfonyl, —C₁₋₆alkyl, and—C₁₋₆alkenyl, or R^(y) and R^(z) may be taken together with the nitrogenof attachment to form an otherwise aliphatic hydrocarbon ring, said ringbeing optionally substituted and having 4 to 7 members, optionallyhaving one carbon replaced with >O, ═N, >NH, or >N(C₁₋₄alkyl), andoptionally substituted Ar wherein said Ar is aryl or heteroaryl.
 2. Thecompound of claim 1, wherein X is

and each R² is independently hydrogen, halo, hydroxyl, methyl, or CF₃.3. The compound of claim 1, wherein X is

and each R² is independently hydrogen, halo, hydroxyl, methyl, or CF₃.4. The compound of claim 1, wherein X is

R² is hydrogen, halo, hydroxyl, methyl, or CF₃, each Z is independentlyC or N wherein R³ is hydrogen, halo, hydroxyl, methyl, or CF₃ when Z isC and R³ is a lone electron pair when Z is N.
 5. The compound of claim1, wherein X is

R² is hydrogen, halo, hydroxyl, methyl, or CF₃, each Z is independentlyC or N wherein R³ is hydrogen, halo, hydroxyl, methyl, or CF₃ when Z isC and R³ is a lone electron pair when Z is N.
 6. The compound of claim 1wherein A is a methlyene optionally substituted with hydrogen,hydroxymethyl, or —C₁₋₄alkyl.
 7. The compound of claim 1, wherein A is abond.
 8. The compound of claim 1, wherein Z is C.
 9. The compound ofclaim 1 wherein R¹ is selected from the group consisting of phenyl,benzyl, quinolinyl, napthalenyl, dichlorophenyl, hydroxyphenyl,trifluoromethylphenyl, benzylamine-, benzyloxy-, benzamide-,chlorobenzyloxy phenyl, benzyloxyphenyl, phenoxy phenyl, benzamidehaving a ring member as a point of attachment and a benzyl substitutenton its N, propyloxy phenyl, and benzenesulfonylamine.
 10. The compoundof claim 1, wherein R¹ is selected from hydrogen, methyl, Br, CI, F,CF₃, methyl, methoxy, OCF₃, Ar, and alkoxy, cycloalkoxy, aryloxy,piperidino, pyrrolidino, and morpholino.
 11. A compound selected fromthe group consisting of:1-(1H-Benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid;1-(5,6-Dichloro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid;1-(5-Trifluoromethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid;1-(5-Chloro-6-fluoro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid; 1-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid; 1-(5-Bromo-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid;1-(5-Methoxy-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid;1-(4-Chloro-6-trifluoromethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid; 1-(5,6-Dimethoxy-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid; 1-(4,5-Dimethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid;1-(5-Trifluoromethoxy-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid;1-{5-[3-(3-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid;1-{5-[3-(2-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid;1-{5-[3-(4-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid;1-[5-(3-Benzyloxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid;1-[5-(4-Benzyloxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid;1-[5-(3-Trifluoromethyl-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid;1-[5-(3,4-Dichloro-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid;1-(5-Bromo-1H-benzoimidazol-2-yl)-3-trifluoromethyl-1H-pyrazole-4-carboxylicacid;1-(5,6-dichloro-1H-benzoimidazol-2-yl)-3-trifluoromethyl-1H-pyrazole-4-carboxylicacid;1-(5-Bromo-1H-benzoimidazol-2-yl)-3,5-dimethyl-1H-pyrazole-4-carboxylicacid;1-(5,6-Dichloro-1H-benzoimidazol-2-yl)-3,5-dimethyl-1H-pyrazole-4-carboxylicacid;1-[5-(4-Hydroxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid;1-[5-(3-Hydroxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid; 1-(5-Chloro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid;1-(5-Bromo-6,7-dimethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid; and pharmaceutically acceptable salts thereof.
 12. Apharmaceutical composition comprising: (a) an effective amount of achemical entity selected from a compound of claim 1, a pharmaceuticallyacceptable salt of said compound, a pharmaceutically acceptable prodrugof said compound, and a pharmaceutically active metabolite of saidcompound; and (b) a pharmaceutically acceptable excipient.
 13. Apharmaceutical composition comprising one or more compounds selectedfrom the group consisting of:1-(1H-Benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid;1-(5,6-Dichloro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid;1-(5-Trifluoromethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid;1-(5-Chloro-6-fluoro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid; 1-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid; 1-(5-Bromo-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid;1-(5-Methoxy-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid;1-(4-Chloro-6-trifluoromethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid; 1-(5,6-Dimethoxy-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid; 1-(4,5-Dimethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid;1-(5-Trifluoromethoxy-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid;1-{5-[3-(3-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid;1-{5-[3-(2-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid;1-{5-[3-(4-Chloro-benzyloxy)-phenyl]-1H-benzoimidazol-2-yl}-1H-pyrazole-4-carboxylicacid;1-[5-(3-Benzyloxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid;1-[5-(4-Benzyloxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid;1-[5-(3-Trifluoromethyl-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid;1-[5-(3,4-Dichloro-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid;1-(5-Bromo-1H-benzoimidazol-2-yl)-3-trifluoromethyl-1H-pyrazole-4-carboxylicacid;1-(5,6-dichloro-1H-benzoimidazol-2-yl)-3-trifluoromethyl-1H-pyrazole-4-carboxylicacid;1-(5-Bromo-1H-benzoimidazol-2-yl)-3,5-dimethyl-1H-pyrazole-4-carboxylicacid;1-(5,6-Dichloro-1H-benzoimidazol-2-yl)-3,5-dimethyl-1H-pyrazole-4-carboxylicacid;1-[5-(4-Hydroxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid;1-[5-(3-Hydroxy-phenyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylicacid; 1-(5-Chloro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid;1-(5-Bromo-6,7-dimethyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid; and pharmaceutically acceptable salts thereof.
 14. A compound ofthe formula:

or a pharmaceutically acceptable salt, pharmaceutically acceptableprodrug, or pharmaceutically active metabolite thereof, wherein each Zis independently C or N; n is 0-4; and R¹ is independently selected fromhydrogen, halogen, nitro, —C₁₋₄alkyl, alkoxy, cycloalkoxy,CON(R^(y))(R^(z)), trifluoromethyl, trifluoromethoxy, N(R^(y))R^(z)(wherein R^(y) and R^(z) are independently hydrogen, aryl, arylsulfonyl,—C₁₋₆alkyl, and —C₁₋₆alkenyl, or R^(y) and R^(z) may be taken togetherwith the nitrogen of attachment to form an otherwise aliphatichydrocarbon ring, said ring being optionally substituted and having 4 to7 members, optionally having one carbon replaced with >O, ═N, >NH, or>N(C₁₋₄alkyl), and optionally substituted Ar wherein said Ar is aryl orheteroaryl; R² is hydrogen, hydroxymethyl or (C₁₋₄)alkyl.
 15. Thecompound of claim 14, wherein each R² is independently hydrogen,hydroxymethyl, or (C₁₋₄)alkyl.
 16. The compound of claim 14, wherein Zis C.
 17. The compound of claim 14, wherein R¹ is selected from thegroup consisting of phenyl, benzyl, quinolinyl, napthalenyl,dichlorophenyl, hydroxyphenyl, trifluoromethylphenyl, benzylamine-,benzyloxy-, benzamide-, chlorobenzyloxy phenyl, benzyloxyphenyl, phenoxyphenyl, benzamide having a ring member as a point of attachment and abenzyl substitutent on its N, propyloxy phenyl, andbenzenesulfonylamine.
 18. The compound of claim 14, wherein R¹ isselected from hydrogen, methyl, Br, CI, F, CF₃, methyl, methoxy, OCF₃,Ar, and alkoxy, cycloalkoxy, aryloxy, piperidino, pyrrolidino, andmorpholino.
 19. A compound selected from the group consisting of:[(1H-Benzoimidazole-2-carbonyl)-amino]-acetic acid;(S)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-propionic acid;[(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;(S)-2-[(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid; [(6-Nitro-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid;(S)-2-[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid; [(5-Iodo-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;[(6-Bromo-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;(S)-2-[(6-Fluoro-1H-benzoimidazole-2-carbonyl)-amino]-propionic acid;(R)-2-[(6-Fluoro-1H-benzoimidazole-2-carbonyl)-amino]-propionic acid;[(6-Methoxy-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;({5-[3-(3-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid;({5-[3-(2-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid;({5-[3-(4-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid;({5-[3-(3-Fluoro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid;({5-[3-(2-Fluoro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid; {[5-(4-Phenoxy-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid;{[5-(3-Benzyloxy-5-fluoro-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid; [(5-Quinolin-3-yl-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid;({5-[3-Chloro-4-(3-chloro-benzyloxy)phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid;({5-[4-(3-Chloro-benzyloxy)-3,5-dimethyl-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid; {[5-(3-Methoxy-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid;{[5-(5-Benzylcarbamoyl-2-fluoro-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid; [(5-Naphthalen-2-yl-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid; {[5-(4-Propoxy-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid;{[5-(4-Chloro-3-methyl-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid;{[5-(5-Chloro-2-fluoro-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid; [(5-Benzoylamino-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;[(6-Benzenesulfonylamino-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid; [(6-Benzylamino-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;(R)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-propionic acid;(R)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-3-hydroxy-propionic acid;(R)-2-[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid;(R)-2-[(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid; (R)-2-[(5-Iodo-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid; and pharmaceutically acceptable salts thereof.
 20. Apharmaceutical composition comprising: (a) an effective amount of achemical entity selected from a compound of claim 14, a pharmaceuticallyacceptable salt of said compound, a pharmaceutically acceptable prodrugof said compound, and a pharmaceutically active metabolite of saidcompound; and (b) a pharmaceutically acceptable excipient.
 21. Apharmaceutical composition comprising one or more compounds selectedfrom the group consisting of:[(1H-Benzoimidazole-2-carbonyl)-amino]-acetic acid;(S)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-propionic acid;[(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;(S)-2-[(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid; [(6-Nitro-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid;(S)-2-[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid; [(5-Iodo-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;[(6-Bromo-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;(S)-2-[(6-Fluoro-1H-benzoimidazole-2-carbonyl)-amino]-propionic acid;(R)-2-[(6-Fluoro-1H-benzoimidazole-2-carbonyl)-amino]-propionic acid;[(6-Methoxy-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;({5-[3-(3-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid;({5-[3-(2-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid;({5-[3-(4-Chloro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid;({5-[3-(3-Fluoro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid;({5-[3-(2-Fluoro-benzyloxy)-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid; {[5-(4-Phenoxy-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid;{[5-(3-Benzyloxy-5-fluoro-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid; [(5-Quinolin-3-yl-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid;({5-[3-Chloro-4-(3-chloro-benzyloxy)phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid;({5-[4-(3-Chloro-benzyloxy)-3,5-dimethyl-phenyl]-1H-benzoimidazole-2-carbonyl}-amino)-aceticacid; {[5-(3-Methoxy-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid;{[5-(5-Benzylcarbamoyl-2-fluoro-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid; [(5-Naphthalen-2-yl-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid; {[5-(4-Propoxy-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid;{[5-(4-Chloro-3-methyl-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid;{[5-(5-Chloro-2-fluoro-phenyl)-1H-benzoimidazole-2-carbonyl]-amino}-aceticacid; [(5-Benzoylamino-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;[(6-Benzenesulfonylamino-1H-benzoimidazole-2-carbonyl)-amino]-aceticacid; [(6-Benzylamino-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid;(R)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-propionic acid;(R)-2-[(1H-Benzoimidazole-2-carbonyl)-amino]-3-hydroxy-propionic acid;(R)-2-[(5,7-Bis-trifluoromethyl-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid;(R)-2-[(5,6-Dichloro-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid; (R)-2-[(5-Iodo-1H-benzoimidazole-2-carbonyl)-amino]-propionicacid; and pharmaceutically acceptable salts thereof.
 22. A method fortreating a hypoxic disorder comprising administering a therapeuticallyeffective amount of a compound of claim 1 or 14 to a patient in needthereof.
 23. The method of claim 22, wherein said hypoxic disorder isanemia.
 24. A method for treating diabetes comprising administering atherapeutically effective amount of a compound of claim 1 or 14 to apatient in need thereof.
 25. A method for wound treatment comprisingadministering a therapeutically effective amount of a compound of claim1 or 14 to a patient in need thereof.
 26. A method for treating ametabolic disorder comprising administering a therapeutically effectiveamount of a compound of claim 1 or 14 to a patient in need thereof. 27.The method of claim 26, wherein said metabolic disorder is obesity ordiabetes.